Antiobese function of platelet-activating factor: increased adiposity in platelet-activating factor receptor-deficient mice with age.

Platelet-activating factor receptor (PAFR)-deficient mice developed a more severe obese state characterized by higher body mass (~25%) and epididymal fat mass (~55%) with age than that of wild-type (WT) littermates. PAFR-deficient mice did not show changes in the expression of critical genes involved in anabolic and catabolic metabolism in adipose, liver, and muscle tissues between 6 and 36 wk. However, a 38-81% reduction in ß3/ß1-adrenergic receptor (AR) and uncoupling protein 1 (UCP1) mRNA and protein levels was observed in the interscapular brown adipose tissue (BAT) of PAFR-deficient mice. Whereas a single injection of the ß3-adrenergic agonist, CL-316,243 (25 µg/kg) increased temperatures in the brown fat and rectums of WT mice, this increase in temperature was markedly suppressed in PAFR-deficient mice. Acetyl-CoA:lyso-platelet-activating factor (PAF) acetyltransferase, which is involved in PAF biosynthesis, and the PAF receptor were predominantly localized in BAT macrophages, whereas brown adipocytes possessed the enzyme and functional PAF receptors. The stimulation of brown adipocytes by PAF induced the expression of ß3-AR mRNA and protein (1.5- and 1.9-fold, respectively), but not that of UCP1. These results indicate that obesity in PAFR-deficient mice resulted from impaired BAT activity and suggest that the antiobese function of PAF occurs through ß3-AR/UCP1 expression in BAT.

Regulation of pregnane X receptor (PXR) function and UGT1A1 gene expression by posttranslational modification of PXR protein.

Human UDP-glucuronosyltransferase (UGT) 1A1 is a critical enzyme responsible for detoxification and metabolism of endogenous and exogenous lipophilic compounds such as bilirubin. The present study shows how cyclin-dependent kinase (CDK) inhibitor roscovitine stimulated the expression of UGT1A1 in HepG2 cells. Pregnane X receptor (PXR)-mediated transactivation of UGT1A1 reporter gene was more prominently enhanced by roscovitine, compared with the basal-, constitutive androstane receptor (CAR)-, and aryl hydrocarbon receptor-mediated activities. We determined the regulatory mechanism of UGT1A1 expression through PXR's stimulation by roscovitine. Although phosphomimetic mutations at Thr290 and Thr408 retained the PXR protein in cytoplasm and attenuated the induction of UGT1A1 expression by both roscovitine and rifampicin, a mutation at Ser350 specifically reduced the activity of PXR induced by roscovitine. Immunoprecipitation analysis revealed that the T290D but not T408D mutant protein remained in cytoplasm by forming a complex with heat shock protein 90 and cytoplasmic CAR retention protein, whereas treatment with proteasome inhibitor MG-132 accumulated the T408D mutant protein in cytoplasm. Transfection with anti-CDK2 small interfering RNA (siRNA) but not anti-CDK1 or CDK5 siRNA led to enhanced expression of UGT1A1. S350D yellow fluorescent protein-PXR fusion protein could translocate from cytoplasm to nucleus similar to the wild-type protein but was detected as an acetylated protein, whose binding with retinoid X receptor (RXR) and histone deacetylase was impaired. Cotransfection with coactivator steroid receptor coactivator (SRC) 2 but not SRC-1 partly recovered its PXR activity. These results indicate that roscovitine stimulated the expression of UGT1A1 by inhibiting CDK2, which phosphorylated PXR at Ser350 to suppress binding with RXR and coactivator and maintain the acetylation of PXR protein.

Up-regulation of TRPM6 transcriptional activity by AP-1 in renal epithelial cells.

Transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of magnesium in the kidney. We recently found that TRPM6 expression is up-regulated by EGF, but the regulatory mechanism has not been clear. TRPM6 mRNA was endogenously expressed in HEK293 cells. TRPM6 mRNA expression was increased by EGF, which was inhibited by U0126, an MEK inhibitor. Promoter activity of human TRPM6 was observed in the TRPM6 5'-flanking region from -1,214 to -718. This promoter activity was enhanced by EGF and inhibited by U0126. Three putative AP-1 binding sites were identified within the region of -1,214/-718. The mutation of the putative AP-1 binding site (-741/-736) completely inhibited the EGF-induced promoter activity. EGF increased p-ERK1/2, c-Fos, c-Jun, and p-c-Jun levels, which were inhibited by U0126. The introduction of c-Fos or c-Jun siRNA inhibited the EGF-induced promoter activity. A chromatin immunoprecipitation assay revealed that c-Fos and c-Jun bind to the AP-1 binding site within the region of -1,214/-718. These results suggest that EGF up-regulates TRPM6 mRNA expression mediate via the activation of ERK/AP-1-dependent pathway.

Induction of UGT1A1 and CYP2B6 by an antimitogenic factor in HepG2 cells is mediated through suppression of cyclin-dependent kinase 2 activity: cell cycle-dependent expression.

Hepatocyte growth factor (HGF), an antimitogenic factor for HepG2 cells, increased mRNA and protein levels of UGT1A1 and CYP2B6, as well as the endogenous cyclin-dependent kinase (CDK) inhibitors p16, p21, and p27 in HepG2 cells but not in HuH6, Caco2, or MCF7 cells. Treatment with 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126) (an extracellular signal-regulated kinase inhibitor) suppressed the HGF-induced expression of UGT1A1 and CYP2B6, as well as p16, p21, and p27 in HepG2 cells. The CDK inhibitor roscovitine also enhanced the expression of UGT1A1, CYP2B6, and CYP3A4. Transfection of anti-CDK2 siRNA led to elevated levels of UGT1A1, CYP2B6, and CYP3A4 in HepG2 and SW480 cells, whereas anti-CDK4 small interfering RNA (siRNA) did not significantly enhance the expression of these enzymes. In fact, CDK2 activity was decreased in HGF-treated HepG2 cells. In cells arrested in S phase by a thymidine block and then released into a synchronous cell cycle, there was a clear dissociation among the activation of CDK2 and the expression of UGT1A1, CYP2B6, and CYP3A4. Furthermore, the induction of CYP3A4 but not UGT1A1 or CYP2B6 mRNA expression by roscovitine was repressed in pregnane X receptor (PXR) siRNA-transfected HepG2 cells. Transfection with constitutive androstane receptor siRNA or PXR siRNA in HepG2 cells did not repress the HGF-stimulated expression of UGT1A1 mRNA. Taken together, our results show that the expression of UGT1A1 and CYP2B6 is negatively regulated through a CDK2 signaling pathway linked to cell cycle progression in HepG2 and SW480 cells, the mechanism of which may differ from that of CYP3A4 expression through PXR phosphorylated by CDK2.

Nutritional status affects fluvastatin-induced hepatotoxicity and myopathy in rats.

Rats that consumed a high-fat and high-sucrose diet (HF diet) developed hepatic steatosis. Treatment of HF diet-fed rats with fluvastatin (8 mg/kg) was lethal, followed by an elevation in levels of plasma aspartate aminotransferase and creatine kinase activities and skeletal muscle toxicity. This study was conducted to determine whether nutritional status affects statin-induced adverse effects in rats. Fluvastatin treatment of rats fed the HF diet led to an increase in systemic exposure, suggesting altered metabolism and elimination. In fact, although hepatic multidrug resistance-associated protein (Mrp) 2 and multidrug resistance (Mdr) 1b protein levels were not significantly changed by fluvastatin treatment for 8 days of rats fed a HF diet, the organic anion-transporting protein (Oatp) 1, Mrp3, CYP1A, CYP2C, UDP-glucuronosyltransferase (UGT) 1A1, and UGT1A5 protein levels were moderately decreased and the Oatp2, CYP3A, and UGT2B1 protein levels were markedly suppressed. No significant difference in the baseline level of Oatp1, Oatp2, Mrp2, Mrp3, Mdr1b, CYP1A, CYP2C, CYP3A, UGT1A1, UGT1A5, or UGT2B1 protein was found between the standard diet- and HF diet-fed groups. In addition, the mRNA levels of Oatp2, CYP2C11, and CYP3A1/2 were markedly decreased in HF diet-fed and fluvastatin-treated rats. There was no significant difference in the glucuronidation activities against fluvastatin among the four groups. In liver cell nuclei, levels of constitutive androstane receptor, pregnane X receptor, and hepatocyte nuclear factor 4α proteins were decreased in fluvastatin-treated HF diet-fed rats, which correlated with the decrease in Oatp2, CYP2C, and CYP3A. Taken together, these results indicate that nutritional status may influence adverse effects of fluvastatin by increasing systemic exposure through modulation of hepatic uptake and elimination.

Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16.

Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption.

Epidermal growth factor increases claudin-4 expression mediated by Sp1 elevation in MDCK cells.

Epidermal growth factor (EGF) increases claudin-4 expression in Madin-Darby canine kidney (MDCK) cells. Here we examined what regulatory mechanisms are involved in the EGF-induced claudin-4 elevation. EGF transiently increased claudin-4 mRNA at 3h and persistently increased its protein for 24h without affecting claudin-1 expression. EGF increased p-ERK1/2 levels, which were inhibited by U0126, a MEK inhibitor. The exogenous expression of constitutively activated MEK increased claudin-4 expression. These results indicate that the activation of ERK1/2 is involved in the EGF-induced claudin-4 elevation. EGF increased Sp1 expression within 1h, which was inhibited by U0126. In immunocytochemistry, Sp1 was distributed in nucleus in control and the EGF-treated cells. The EGF-induced claudin-4 elevation was inhibited by mithramycin, a Sp1 inhibitor, and Sp1 small interfering RNA. We suggest that EGF activates a MEK/ERK pathway and increases Sp1 expression, resulting in an elevation of claudin-4 expression.

Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway.

Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg(2+) in the thick ascending limb of Henle. The tight junctional localization and Mg(2+) transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg(2+). Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg(2+) reabsorption.

TRPM6 expression and cell proliferation are up-regulated by phosphorylation of ERK1/2 in renal epithelial cells.

Transient receptor potential melastatin 6 (TRPM6) is a magnesium channel and expressed in the intestine and renal distal tubules. Little is known about the regulatory mechanism of TRPM6 expression and the role of magnesium influx. EGF increased the phosphorylation of ERK1/2 and TRPM6 expression that were inhibited by U0126 in renal epithelial NRK-52E cells. Furthermore, EGF enhanced the influx of magnesium, whereas U0126 and TRPM6 siRNA inhibited it. EGF increased the proportion of cells in S phase, whereas U0126 and TRPM6 siRNA increased the proportion in G1 phase. The phosphorylation of ERK1/2 may up-regulate TRPM6 expression and magnesium influx, resulting in an increase in cell proliferation with a shift from G1 to S phase.

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells.

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Expression of CAR in SW480 and HepG2 cells during G1 is associated with cell proliferation.

Constitutive androstane receptor (CAR) is a transcription factor to regulate the expression of several genes related to drug-metabolism. Here, we demonstrate that CAR protein accumulates during G1 in human SW480 and HepG2 cells. After the G1/S phase transition, CAR protein levels decreased, and CAR was hardly detected in cells by the late M phase. CAR expression in both cell lines was suppressed by RNA interference-mediated suppression of CDK4. Depletion of CAR by RNA interference in both cells and by hepatocyte growth factor treatment in HepG2 cells resulted in decreased MDM2 expression that led to p21 upregulation and repression of HepG2 cell growth. Thus, our results demonstrate that CAR expression is an early G1 event regulated by CDK4 that contributes to MDM2 expression; these findings suggest that CAR may influence the expression of genes involved in not only the metabolism of endogenous and exogenous substances but also in the cell proliferation.

Expression of hepatic UDP-glucuronosyltransferase 1A1 and 1A6 correlated with increased expression of the nuclear constitutive androstane receptor and peroxisome proliferator-activated receptor alpha 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 alpha (PPARalpha) protein levels were significantly enhanced by intake of the HF1 diet. Additionally, administration of the PPARalpha 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 PPARalpha in liver cell nuclei, and this effect appears to be associated with alterations in UGT1A1 and UGT1A6 expression.

Down-regulation of TRPM6-mediated magnesium influx by cyclosporin A.

Transient receptor potential melastatin 6 (TRPM6) is distributed along the apical membrane of the renal tubular cells and is involved in the reabsorption of magnesium. In this study, we show that TRPM6 expression is suppressed by cyclosporin A (CsA) via a down-regulation of c-Fos expression. TRPM6 was expressed in NRK-52E, but not in Madin-Darby canine kidney cells. In contrast, its homolog, TRPM7, was equally expressed in both cells. In NRK-52E cells, CsA dose-dependently decreased TRPM6 expression without affecting TRPM7 expression. Magnesium load measurements revealed the rise in the intracellular free magnesium concentration ([Mg2+]i) to be inhibited by CsA. The transfection of TRPM6 siRNA decreased TRPM6 expression without affecting TRPM7 expression and inhibited the elevation of [Mg2+]i. CsA did not affect the intracellular distribution of nuclear factor of activated T cells (NFATc). Furthermore, TRPM6 expression was not changed by a NFATc inhibitor. Next, we examined the effect of CsA on the transcription factors c-Fos and c-Jun. CsA decreased c-Fos expression without affecting c-Jun expression. The transfection of c-Fos siRNA suppressed TRPM6 expression without affecting TRPM7 expression. We suggest that CsA decreases TRPM6 expression mediated by inhibition of c-Fos transcription, resulting in a decrease of renal Mg2+ reabsorption.

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 1alpha (HNF1alpha) 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)(7)TAA 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)(7)TAA mutations was still lower. Thus, UGT1A1 gene promoter variations, including the TA repeat polymorphism and T-3279G gtPBREM, have important clinical implications.

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells.

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK(1) cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.

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.

Physicochemical characterization and biological effects of inulin enzymatically synthesized from sucrose.

We first developed the method to produce inulin from sucrose using an enzyme from Bacillus sp. 217C-1. Synthesized inulin consists of a linear polymer having beta(2-1) linkages of d-fructose with one terminal glucose. The synthesized inulin has similar properties (pH and thermal stability, Maillard reaction, and in vitro fermentation) to plant-derived inulin. The marked difference is the polydispersity of the inulin chain length. Synthesized inulin with a narrow degree range of fructose polymerization shows better solubility in water than plant-derived inulin. Synthesized inulin (5%, w/w) treatment for 12 weeks reduced the elevation in body weight and serum and liver lipids in rats fed high fat- and high sucrose-supplemented diets, and blood glucose in rats fed a standard diet. Synthesized inulin (15%, w/w) significantly suppressed the elevation in blood glucose of human healthy subjects after dextrin loading. These results suggest that daily intake of synthesized inulin modulates carbohydrate and lipid metabolism.

A platelet-activating factor (PAF) receptor deficiency exacerbates diet-induced obesity but PAF/PAF receptor signaling does not contribute to the development of obesity-induced chronic inflammation.

Platelet-activating factor (PAF) is a well-known phospholipid that mediates acute inflammatory responses. In the present study, we investigated whether PAF/PAF receptor signaling contributed to chronic inflammation in the white adipose tissue (WAT) of PAF receptor-knockout (PAFR-KO) mice. Body and epididymal WAT weights were higher in PAFR-KO mice fed a high-fat diet (HFD) than in wild-type (WT) mice. TNF-α mRNA expression levels in epididymal WAT and the infiltration of CD11c-positive macrophages into epididymal WAT, which led to chronic inflammation, were also elevated in HFD-fed PAFR-KO mice. HFD-fed PAFR-KO mice had higher levels of fasting serum glucose than HFD-fed WT mice as well as impaired glucose tolerance. Although PAF receptor signaling up-regulated the expression of TNF-α and lipopolysaccharide induced the expression of acyl-CoA:lysophosphatidylcholine acyltransferase 2 (LPCAT2) mRNA in bone marrow-derived macrophages, no significant differences were observed in the expression of LPCAT2 mRNA and PAF levels in epididymal WAT between HFD-fed mice and normal diet-fed mice. In addition to our previous finding in which energy expenditure in PAF receptor (PAFR)-deficient mice was low due to impaired brown adipose tissue function, the present study demonstrated that PAF/PAF receptor signaling up-regulated the expression of Ucp1 mRNA, which is essential for cellular thermogenesis, in 3T3-L1 adipocytes. We concluded that the marked accumulation of abdominal fat due to HFD feeding led to more severe chronic inflammation in WAT, which is associated with glucose metabolism disorders, in PAFR-KO mice than in WT mice, and PAF/PAF receptor signaling may regulate energy expenditure and adiposity.

Role of inducible nitric oxide synthase in skeletal adaptation to acute increases in mechanical loading.

To clarify the role of nitric oxide (NO) in regulation of bone metabolism in response to skeletal loading, we examined inducible NO synthase (iNOS) gene knockout mice in the tail-suspension model. Histomorphometric analyses of proximal tibias revealed that 7 days of tail suspension decreased the bone volume (BV/TV) and bone formation rate (BFR/BS) and increased the osteoclast surface (Oc.S/BS) in mice with all iNOS genotypes. Both iNOS+/+ and iNOS+/- mice responded to subsequent 14-day reloading, with increases in BV/TV and BFR/BS and a decrease in Oc.S/BS, whereas these responses were abolished in iNOS-/- mice. The osteoblasts flattened after tail suspension appeared cuboidal during subsequent reloading. Immunoreactivity for iNOS was detected in these osteoblasts and osteocytes by immunohistochemistry. These defective responses after reloading were rescued in iNOS-/- mice by treatment with an NO donor nitroglycerine (NG). Conversely, the responses in iNOS+/+ mice were inhibited by treatment with an NOS inhibitor aminoguanidine (AG). In bone marrow cell cultures, mineralized nodules derived from iNOS-/- mice after reloading were significantly reduced. Taken together, our results suggest that NO generated by iNOS in osteoblasts plays a critical role in adjusting bone turnover and increasing osteogenic activity in response to the acute increase in mechanical loading after tail suspension.

The induction of human UDP-glucuronosyltransferase 1A1 mediated through a distal enhancer module by flavonoids and xenobiotics.

We identified the UDP-glucuronosyltransferase (UGT) 1A1 5'-upstream region that confers UGT1A1 induction by various agents, including flavonoids, on a luciferase reporter gene and has the properties of a transcriptional enhancer. Chrysin- and rifampicin-response activities were traced to the same element as a 290-bp distal enhancer module (-3483/-3194), in which the reporter activities were enhanced by activators of nuclear receptors [constitutive androstane receptor (CAR) and pregnane X receptor (PXR)] and transcription factor [aryl hydrocarbon receptor (AhR)]. Utilizing transactivation experiments with the UGT1A1 290-bp reporter gene, we assessed UGT1A1 induction by various flavonoids. 5,7-Dihydroxyflavones with varying substituents in the B-ring and gallocatechin dimers increased the reporter activity in a time- and dose-dependent manner. The treatment of HepG2 cells with the flavonoids for 24 hr elevated the expression of mRNAs and proteins of UGT1A1 and CYP1A1, while the mRNA levels of CYP2B6, CYP3A4, CAR, PXR and AhR was not altered. Chrysin and rifampicin induced the activation of the wild-type reporter gene and T-3263G-mutated gene to a similar extent in HepG2 cells cotransfected with expression vectors of CAR and PXR. Mutation of the AhR core binding region most prominently suppressed the activation of the 290-bp reporter gene by chrysin and baicalein, while mutations of four putative nuclear receptor motifs (DR4 element, PXRE, CARE and DR3 element) partly decreased its activation. Taken together, the results indicate that UGT1A1 was induced in response to flavonoids and xenobiotics through the transactivation of the 290-bp reporter gene, that was a multi-component enhancer containing CAR, PXR and AhR motifs.