Reduction in cytochrome P-450 enzyme expression is associated with repression of CAR (constitutive androstane receptor) and PXR (pregnane X receptor) in mouse liver during the acute phase response

Expression of P-450 (Cyp) enzymes is reduced in liver during the acute phase response, contributing to the decrease in bile acid levels and drug metabolism during infection. Nuclear hormone receptors CAR and PXR are key transactivators of Cyp2b and Cyp3a genes, respectively. Injection of bacterial lipopolysaccharide (LPS) induced the expected reduction in Cyp2b10 and Cyp3a mRNA levels in mouse liver. These decreases were associated with a marked reduction in CAR and PXR mRNA levels within 4 h following treatment. LPS-induced CAR and PXR repression were dose-dependent and sustained for at least 16 h. LPS treatment also reversed the up-regulation of Cyp3a in mice pre-treated with PXR ligand RU486. In addition, we observed a concomitant decrease in RXR (retinoid X receptor) mRNA levels, the obligatory partner of both CAR and PXR for high affinity binding to DNA. These findings represent one possible molecular mechanism underlying sepsis-induced repression of Cyp enzymes.     

Modulation of Cyp3a11 mRNA expression by alpha-tocopherol but not gamma-tocotrienol in mice

Metabolism of vitamin E is initiated by cytochrome P450 (CYP) enzymes usually involved in the metabolism of xenobiotics. Like other CYP substrates, vitamin E induced a reporter gene under the control of the pregnane X receptor (PXR) which regulates the expression of CYPs including CYP3A4. gamma-Tocotrienol, the most effective PXR activator, also induced endogenous CYP3A4 mRNA in HepG2 cells. Since these findings imply an interference of vitamin E with drug metabolism it was deemed necessary to investigate their in vivo relevance. Therefore, mice were grown for 3 months with alpha-tocopherol-deficient, -adequate, and -supranutritional diet, i.e. 2, 20 and 200 mg RRR-alpha-tocopheryl acetate/kg diet, respectively. Half of them received 250 microg gamma-tocotrienol/day for the last 7 days. After 3 months, hepatic levels of Cyp3a11 mRNA, the murine homolog to human CYP3A4, were about 2.5-fold higher in the 20 and 200 mg alpha-tocopherol groups than in the 2 mg group. After feeding 200 mg alpha-tocopherol for 9 months, Cyp3a11 mRNA was 1.7-fold higher than after 3 months. In contrast, gamma-tocotrienol did not induce Cyp3a11 mRNA. This could be explained by its high metabolism as demonstrated by the 20- to 25-fold increase in the urinary excretion of gamma-CEHC, the final metabolite of gamma-tocotrienol degradation. In conclusion, alpha-tocopherol maintains an adequate level of xenobiotic-metabolizing enzymes. If fed in supranutritional dosages, especially for longer times, alpha-tocopherol induces Cyp3a11 to levels which might interfere with drug metabolism.     

Regulation of reticuloendothelial iron transporter MTP1 (Slc11a3) by inflammation

Acute and chronic inflammation cause many changes in total body iron metabolism including the sequestration of iron in phagocytic cells of the reticuloendothelial system. This change in iron metabolism contributes to the development of the anemia of inflammation. MTP1, the duodenal enterocyte basolateral iron exporter, is also expressed in the cells of the reticuloendothelial system (RES) and is likely to be involved in iron recycling of these cells. In this study, we use a lipopolysaccharide model of the acute inflammation in the mouse and demonstrate that MTP1 expression in RES cells of the spleen, liver, and bone marrow is down-regulated by inflammation. The down-regulation of splenic expression of MTP1 by inflammation was also observed in a Leishmania donovani model of chronic infection. The response of MTP1 to lipopolysaccharide (LPS) requires signaling through the LPS receptor, Toll-like receptor 4 (TLR4). In mice lacking TLR4, MTP1 expression is not altered in response to LPS. In addition, mice lacking tumor necrosis factor-receptor 1a respond appropriately to LPS with down-regulation of MTP1, despite hyporesponsiveness to tumor necrosis factor-alpha signaling, suggesting that this cytokine may not be required for the LPS effect. We hypothesize that the iron sequestration in the RES system that accompanies inflammation is because of down-regulation of MTP1.     

Regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase SHP-1

The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.     

Coordinated induction of bile acid detoxification and alternative elimination in mice: role of FXR-regulated organic solute transporter-alpha/beta in the adaptive response to bile acids

The bile acid receptor farnesoid X receptor (FXR) is a key regulator of hepatic defense mechanisms against bile acids. A comprehensive study addressing the role of FXR in the coordinated regulation of adaptive mechanisms including biosynthesis, metabolism, and alternative export together with their functional significance is lacking. We therefore fed FXR knockout (FXR(-/-)) mice with cholic acid (CA) and ursodeoxycholic acid (UDCA). Bile acid synthesis and hydroxylation were assessed by real-time RT-PCR for cytochrome P-450 (Cyp)7a1, Cyp3a11, and Cyp2b10 and mass spectrometry-gas chromatography for determination of bile acid composition. Expression of the export systems multidrug resistance proteins (Mrp)4-6 in the liver and kidney and the recently identified basoalteral bile acid transporter, organic solute transporter (Ost-alpha/Ost-beta), in the liver, kidney, and intestine was also investigated. CA and UDCA repressed Cyp7a1 in FXR(+/+) mice and to lesser extents in FXR(-/-) mice and induced Cyp3a11 and Cyp2b10 independent of FXR. CA and UDCA were hydroxylated in both genotypes. CA induced Ost-alpha/Ost-beta in the liver, kidney, and ileum in FXR(+/+) but not FXR(-/-) mice, whereas UDCA had only minor effects. Mrp4 induction in the liver and kidney correlated with bile acid levels and was observed in UDCA-fed and CA-fed FXR(-/-) animals but not in CA-fed FXR(+/+) animals. Mrp5/6 remained unaffected by bile acid treatment. In conclusion, we identified Ost-alpha/Ost-beta as a novel FXR target. Absent Ost-alpha/Ost-beta induction in CA-fed FXR(-/-) animals may contribute to increased liver injury in these animals. The induction of bile acid hydroxylation and Mrp4 was independent of FXR but could not counteract liver toxicity sufficiently. Limited effects of UDCA on Ost-alpha/Ost-beta may jeopardize its therapeutic efficacy.     

Vitamin D and the regulation of placental inflammation

The vitamin D-activating enzyme 1α-hydroxylase (CYP27B1) and vitamin D receptor (VDR) support anti-inflammatory responses to vitamin D in many tissues. Given the high basal expression of CYP27B1 and VDR in trophoblastic cells from the placenta, we hypothesized that anti-inflammatory effects of vitamin D may be particularly important in this organ. Pregnant wild type (WT) mice i.p. injected with LPS showed elevated expression of mouse Cyp27b1 (4-fold) and VDR (6-fold). Similar results were also obtained after ex vivo treatment of WT placentas with LPS. To assess the functional impact of this, we carried out ex vivo studies using placentas -/- for fetal (trophoblastic) Cyp27b1 or VDR. Vehicle-treated -/- placentas showed increased expression of IFN-γ and decreased expression of IL-10 relative to +/+ placentas. LPS-treated -/- placentas showed increased expression of TLR2, IFN-γ, and IL-6. Array analyses identified other inflammatory factors that are dysregulated in Cyp27b1(-/-) versus Cyp27b1(+/+) placentas after LPS challenge. Data highlighted enhanced expression of IL-4, IL-15, and IL-18, as well as several chemokines and their receptors, in Cyp27b1(-/-) placentas. Similar results for IL-6 expression were observed with placentas -/- for trophoblastic VDR. Finally, ex vivo treatment of WT placentas with the substrate for Cyp27b1, 25-hydroxyvitamin D(3), suppressed LPS-induced expression of IL-6 and the chemokine Ccl11. These data indicate that fetal (trophoblastic) vitamin D plays a pivotal role in controlling placental inflammation. In humans, this may be a key factor in placental responses to infection and associated adverse outcomes of pregnancy.     

Regulation of gene expression of hepatic drug metabolizing enzymes and transporters by the Toll-like receptor 2 ligand, lipoteichoic acid

Expression of hepatic drug metabolizing enzymes (DMEs) is altered in infection and inflammation. However, the role of Gram+ve bacterial components and their receptor, Toll-like receptor (TLR) 2 in regulation of hepatic DMEs is unknown. Gene expression of DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRα). The TLR2 ligand, lipoteichoic acid (LTA) reduced RNA levels of CAR and its target genes, Cyp2b10, Cyp2a4 and Sultn in mouse liver (∼60-80% reduction). Hepatic genes regulated by PXR and CAR, Cyp3a11 and Mrp2 were moderately reduced by LTA, along with ∼50% reduction of PXR RNA and nuclear protein levels of RXRα. The effects of LTA were significantly attenuated by pre-treatment with the Kupffer cell inhibitor, gadolinium chloride, indicating that Kupffer cells contribute to LTA-mediated down-regulation of hepatic genes. These results indicate that treatment with Gram+ve bacterial components preferentially down-regulate CAR and its target genes in the liver.     

TLR4 Expression by Liver Resident Cells Mediates the Development of Glucose Intolerance and Insulin Resistance in Experimental Periodontitis

Background

Results from epidemiological studies indicate a close association between periodontitis and type 2 diabetes mellitus. However, the mechanism linking periodontitis to glucose intolerance (GI) and insulin resistance (IR) is unknown. We therefore tested the hypothesis that periodontitis induces the development of GI/IR through a liver Toll-like receptor 4 (TLR4) dependent mechanism.

Methods

TLR4 chimeric mice were developed by bone marrow transplantation using green fluorescent protein expressing TLR4WT mouse (GFPWT) as donor and TLR4 WT or TLR4-/- as recipient mice (GFPWT:WT and GFPWT:KO chimeras respectively). These chimeras were subjected to experimental chronic periodontitis induced by repeated applications of LPS to the gingival sulci for 18 weeks. The levels of GI/IR were monitored and plasma cytokines and LPS were determined at 18 weeks when differences in glucose tolerance were most apparent. Cytokine gene expression was measured in liver tissue by qPCR.

Results

Alveolar bone loss was significantly greater in GFPWT:WT chimeras treated with LPS compared with chimeras treated with PBS or GFPWT:KO chimeras. However, the degree of gingival inflammation was similar between GFPWT:WT and GFPWT:KO mice with LPS application. Severe GI/IR occurred in GFPWT:WT chimeras but not in the GFPWT:KO chimeras that were subjected to 18 weeks of LPS. Serum LPS was detected only in animals to which LPS was applied and the level was similar in GFPWT:WT and GFPWT:KO mice at the 18 week time point. Surprisingly, there was no significant difference in the plasma levels of IL1β, IL6 and TNFα at 18 weeks in spite of the severe GI/IR in the GFPWT:WT chimeras with LPS application. Also, no difference in the expression of TNFα or IL6 mRNA was detected in the liver of GFPWT:WT vs GFPWT:KO mice. In contrast, liver IL1β expression was significantly greater in GFPWT:WT chimeras compared to GFPWT:KO chimeras treated with LPS.

Conclusion

We observed that GFPWT:WT, but not GFPWT:KO chimeras, treated with LPS developed GI/IR despite similar degrees of gingival inflammation, circulating cytokine levels, and LPS concentrations. We conclude that LPS from periodontitis sites has a pivotal role in triggering the development of GI/IR through a mechanism that involves TLR4 expression by resident macrophages/Kupffer cells in the liver.     

Low TLR4 expression by liver dendritic cells correlates with reduced capacity to activate allogeneic T cells in response to endotoxin

Signaling via TLRs results in dendritic cell (DC) activation/maturation and plays a critical role in the outcome of primary immune responses. So far, no data exist concerning TLR expression by liver DC, generally regarded as less immunostimulatory than secondary lymphoid tissue DC. Because the liver lies directly downstream from the gut, it is constantly exposed to bacterial LPS, a TLR4 ligand. We examined TLR4 expression by freshly isolated, flow-sorted C57BL/10 mouse liver DC compared with spleen DC. Real-time PCR revealed that liver CD11c+CD8alpha- (myeloid) and CD11c+CD8alpha+ ("lymphoid-related") DC expressed lower TLR4 mRNA compared with their splenic counterparts. Lower TLR4 expression correlated with reduced capacity of LPS (10 ng/ml) but not anti-CD40-stimulated liver DC to induce naive allogeneic (C3H/HeJ) T cell proliferation. By contrast to LPS-stimulated splenic DC, these LPS-activated hepatic DC induced alloantigen-specific T cell hyporesponsiveness in vitro, correlated with deficient Th1 (IFN-gamma) and Th2 (IL-4) responses. When higher LPS concentrations (> or =100 ng/ml) were tested, the capacity of liver DC to induce proliferation of T cells and Th1-type responses was enhanced, but remained inferior to that of splenic DC. Hepatic DC activated by LPS in vivo were inferior allogeneic T cell stimulators compared with splenic DC, whereas adoptive transfer of LPS-stimulated (10 ng/ml) liver DC induced skewing toward Th2 responses. These data suggest that comparatively low expression of TLR4 by liver DC may limit their response to specific ligands, resulting in reduced or altered activation of hepatic adaptive immune responses.     

Beta2-integrin-induced p38 MAPK activation is a key mediator in the CD14/TLR4/MD2-dependent uptake of lipopolysaccharide by hepatocytes

The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the beta2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.     

Behavioral effects of inhibition of cannabinoid metabolism: The amidase inhibitor AM374 enhances the suppression of lever pressing produced by exogenously administered anandamide

Biochemical investigations have identified putative enzymatic pathways for the synthesis and metabolism of endogenous cannabinoids. Anandamide amidase is an enzyme that metabolizes anandamide into arachadonic acid and ethanolamine. Using in vitro methods, various inhibitors of amidase have been identified. The present studies were undertaken to determine if the amidase inhibitor AM 374 could enhance the effects of intraperitoneal (IP) injections of anandamide. Three studies were conducted to investigate the effects of various drug treatments on fixed ratio 5 operant lever pressing for food reinforcement. In the first study, the effects of different doses of anandamide were assessed, and it was demonstrated that 5.0 and 10.0 mg/kg anandamide IP significantly suppressed lever pressing, while 2.5 mg/kg produced very little effect. The second study tested the effects of intraventricular (ICV) injections of AM 374, and it was observed that doses up to 10.0, 20.0 and 40 microg AM 374 had no significant effect upon lever pressing. The third study investigated the combined effect of AM374 with a low dose of anandamide. Rats received two drug injections: one ICV and one IP. Four different drug treatments were assessed: 1) ICV vehicle + IP vehicle, 2) ICV vehicle + 2.5 mg/kg anandamide IP, 3) ICV 20.0 microg AM 374 + IP vehicle, and 4) ICV 20 microg AM 374 + 2.5 mg/kg anandamide IP. Combined administration of AM 374 plus anandamide led to a significant decrease in lever pressing compared to either AM374 or anandamide administered alone. Observations of the animals treated with the combination of AM374 plus anandamide indicated that the drug combination resulted in motor slowing, which is consistent with the notion that stimulation of cannabinoid receptors produced a motor deficit that interfered with lever pressing. Although AM374 produced no effect on its own, this amidase inhibitor did enhance the behavioral effect of a low dose of anandamide. These results are consistent with the notion that AM 374 inhibited the enzymatic breakdown of exogenously injected anandamide. This type of procedure can be used to assess a variety of different compounds for their ability to inhibit cannabinoid metabolism.     

Milano Summer Particulate Matter (PM10) Triggers Lung Inflammation and Extra Pulmonary Adverse Events in Mice

Recent studies have suggested a link between particulate matter (PM) exposure and increased mortality and morbidity associated with pulmonary and cardiovascular diseases; accumulating evidences point to a new role for air pollution in CNS diseases. The purpose of our study is to investigate PM10sum effects on lungs and extra pulmonary tissues. Milano PM10sum has been intratracheally instilled into BALB/c mice. Broncho Alveolar Lavage fluid, lung parenchyma, heart and brain were screened for markers of inflammation (cell counts, cytokines, ET-1, HO-1, MPO, iNOS), cytotoxicity (LDH, ALP, Hsp70, Caspase8-p18, Caspase3-p17) for a putative pro-carcinogenic marker (Cyp1B1) and for TLR4 pathway activation. Brain was also investigated for CD68, TNF-α, GFAP. In blood, cell counts were performed while plasma was screened for endothelial activation (sP-selectin, ET-1) and for inflammation markers (TNF-α, MIP-2, IL-1β, MPO). Genes up-regulation (HMOX1, Cyp1B1, IL-1β, MIP-2, MPO) and miR-21 have been investigated in lungs and blood. Inflammation in the respiratory tract of PM10sum-treated mice has been confirmed in BALf and lung parenchyma by increased PMNs percentage, increased ET-1, MPO and cytokines levels. A systemic spreading of lung inflammation in PM10sum-treated mice has been related to the increased blood total cell count and neutrophils percentage, as well as to increased blood MPO. The blood-endothelium interface activation has been confirmed by significant increases of plasma ET-1 and sP-selectin. Furthermore PM10sum induced heart endothelial activation and PAHs metabolism, proved by increased ET-1 and Cyp1B1 levels. Moreover, PM10sum causes an increase in brain HO-1 and ET-1. These results state the translocation of inflammation mediators, ultrafine particles, LPS, metals associated to PM10sum, from lungs to bloodstream, thus triggering a systemic reaction, mainly involving heart and brain. Our results provided additional insight into the toxicity of PM10sum and could facilitate shedding light on mechanisms underlying the development of urban air pollution related diseases.     

Agonistic antibody to TLR4/MD-2 protects mice from acute lethal hepatitis induced by TNF-alpha

LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-alpha, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-kappaB through TLR4/MD-2.     

S‐allyl cysteine,an active ingredient of garlic,attenuates acute liver dysfunction induced by lipopolysaccharide/ d‐galactosamine in mouse: Underlying mechanisms

In the present study, beneficial effect of S‐allyl cysteine (SAC) was evaluated in the lipopolysaccharide/d ‐galactosamine (LPS/d ‐Gal) model of acute liver injury (ALI). To mimic ALI, LPS and d ‐Gal (50 μg/kg and 400 mg/kg, respectively) were intraperitoneally administered and animals received SAC per os (25 or 100 mg/kg/d) for 3 days till 1 hour before LPS/d ‐Gal injection. Pretreatment of LPS/d ‐Gal group with SAC‐lowered activities of alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase and partially reversed inappropriate alterations of hepatic oxidative stress‐ and inflammation‐related biomarkers including liver reactive oxygen species, malondialdehyde, and hepatic activity of the defensive enzyme superoxide dismutase, ferric reducing antioxidant power (FRAP), toll‐like receptor‐4 (TLR4), cyclooxygenase 2, NLR family pyrin domain containing 3 (NLRP3), caspase 1, nuclear factor κB (NF‐κB), interleukin 1β (IL‐1β), IL‐6, tumor necrosis factor‐α, and myeloperoxidase activity. Additionally, SAC was capable to ameliorate apoptotic biomarkers including caspase 3 and DNA fragmentation. In summary, SAC can protect liver against LPS/d ‐Gal by attenuation of neutrophil infiltration, oxidative stress, inflammation, apoptosis, and pyroptosis which is partly linked to its suppression of TLR4/NF‐κB/NLRP3 signaling.     

Thermoregulatory responses of rats to conventional preparations of lipopolysaccharide are caused by lipopolysaccharide per se-- not by lipoprotein contaminants

LPS preparations cause a variety of body temperature (T(b)) responses: monophasic fever, different phases of polyphasic fever, and hypothermia. Conventional (c) LPS preparations contain highly active lipoprotein contaminants (endotoxin proteins). Whereas LPS signals predominantly via the Toll-like receptor (TLR) 4, endotoxin proteins signal via TLR2. Several TLR2-dependent responses of immunocytes to cLPS in vitro are triggered by endotoxin proteins and not by LPS itself. We tested whether any T(b) response to cLPS from Escherichia coli 055:B5 is triggered by non-TLR4-signaling contaminants. A decontaminated (d) LPS preparation (free of endotoxin proteins) was produced by subjecting cLPS to phenol-water reextraction. The presence of non-TLR4-signaling contaminants in cLPS (and their absence in dLPS) was confirmed by showing that cLPS (but not dLPS) induced IL-1beta expression in the spleen and increased serum levels of TNF-alpha and IL-1beta of C3H/HeJ mice; these mice bear a nonfunctional TLR4. Yet, both cLPS and dLPS caused cytokine responses in C3H/HeOuJ mice; these mice bear a fully functional TLR4. We then studied the T(b) responses to cLPS and dLPS in Wistar rats preimplanted with jugular catheters. At a neutral ambient temperature (30 degrees C), a low (0.1 microg/kg iv) dose of cLPS caused a monophasic fever, whereas a moderate (10 microg/kg iv) dose produced a polyphasic fever. In the cold (20 degrees C), a high (500 microg/kg iv) dose of cLPS caused hypothermia. All T(b) responses to dLPS were identical to those of cLPS. We conclude that all known T(b) responses to LPS preparations are triggered by LPS per se and not by non-TLR4-signaling contaminants of such preparations.     

Cytochrome P-450 2E1 in Rat Liver Peroxisomes: Downregulation by Ischemia/Reperfusion-Induced Oxidative Stress

Cytochrome P-450 containing enzymes, known to be present in the endoplasmic reticulum and mitochondria, catalyze the oxidation of various compounds. In this study we have used highly purified peroxisomes (>95%) to provide evidence by analytical cell fractionation, enzyme activity, Western blot, and immunocytochemical analysis that cytochrome P-450 2E1 (Cyp 2E1) is present in peroxisomes. Similar specific activities of aniline hydroxylase, a Cyp 2E1-dependent enzyme, in purified peroxisomes (0.72 ± 0.03 nmol/min/mg protein) and microsomes (0.58 ± 0.03 nmol/min/mg protein) supports the conclusion that peroxisomes contain significant amount of Cyp 2E1. This peroxisomal Cyp 2E1 was also induced in acetone-treated rat liver. The status of microsomal and peroxisomal Cyp 2E1 was also examined following ischemia/reperfusion-induced oxidative stress. Ischemia alone had no effect; however, reperfusion following ischemia resulted in decrease in Cyp 2E1 both in microsomes and peroxisomes. This demonstration of cytochrome P-450 2E1 in peroxisomes and its downregulation during ischemia/reperfusion describes a new role for this organelle in cytochrome P-450 related cellular metabolism and in oxidative stress induced disease conditions.     

Effects of centrally injected amylin on sexually behavior of male rats.

Amylin (AMY) is a peptide of pancreatic origin principally involved in the carbohydrate metabolism, but that may interfere with central and peripheral dopamine (DA) pathways. The peptide, injected intracerebroventricularly (ICV) at the dose of 2.5 microg/rat, induced a decrease of copulatory activity in good copulators (GCO) and sluggish (SLU) male rats. The dose of 0.1 microg/rat did not affect significantly the sexual behavior of GCO rats, whereas AMY 0.5 microg/rat increased only the latency and reduced the frequency of ejaculation. At the dose of 2.5 microg/rat AMY antagonized the activation of sexual behavior induced by the DA receptor agonist, apomorphine administered subcutaneously (SC) at the dose of 100 microg/kg. Moreover, this inhibitory effect was blocked by the calcitonin gene-related peptide and AMY receptor antagonist, CGRP (8-37) fragment (injected ICV at the dose of 1 microg/rat). These data suggest that AMY may exert inhibitory effects on male sexual behavior in rats, probably interfering with central DA neurotransmission and with CGRP receptors.     

Regulation of SLC11A3 by inflammation

Acute and chronic inflammatory states are characterized by changes in body iron metabolism. These changes include a drop in serum iron, an increase in the rate of plasma iron disappearance, a decline in the rate of plasma iron turnover, reticuloendothelial system (RES) cell iron sequestration and a decline in intestinal iron absorption. This response is elicited by a variety of metabolic conditions and acute bacterial infections, especially gram-negative bacteria, and by experimental mediators of inflammation such as endotoxin and turpentine. These changes in iron metabolism contribute to the development of the anemia of chronic diseases. SLC11A3 (aka MTP1, ferroportin 1, IREG1) is a metal transporter that exports iron from the cytosol of cells and was initially identified as the duodenal epithelial basolateral iron transporter. Recent identification of a MTP1 mutation leading to hemochromatosis in man adds further weight to the hypothesis that MTP1 is involved in iron homeostasis. RES cells are responsible for the recycling of iron from the breakdown of heme from senescent erythrocytes and MTP1 has been hypothesized to be the key iron exporter in these cells. Supporting this hypothesis is the observation that MTP1 is expressed in the RES macrophages of the spleen, Kupffer cells, bone marrow and lymph node histiocytes, mesangial cells, brain microglial cells. In a mouse (C57/Bl6) model of lipopolysaccharide (LPS) induced acute inflammation, MTP1 expression in the cells of the RES is regulated by acute inflammation. Immunohistochemical staining of tissues, using an anti-MTP1 antibody, of mice given parenteral injections of LPS demonstrated down-regulation of MTP1 expression in the RES cells of the spleen and liver and also in the duodenal epithelial cells compared to control animals. Western blotting of total liver and spleen lysates confirmed the decline in MTP1 protein expression induced by LPS. In addition, RT-PCR analysis showed that LPS treatment also resulted in a decline in MTP1 mRNA in spleen, liver and duodenum compared to controls. One clue to the molecular signaling mechanism for MTP1 down-regulation by LPS comes from the study of the C3H/HeJ mouse, which lacks a functional LPS receptor, toll-like receptor 4 (TLR4). C3H/HeJ mice are resistant to the toxic and hypoferraemic effects of LPS. Similarly, a down-regulation of MTP1 in response to LPS in the C3H/HeJ mice was not observed. This finding indicates that the down-regulation of MTP1 by LPS requires signaling through TLR4. Despite resistance to LPS, treatment of C3H/HeJ mice with turpentine, an inducer of sterile inflammation, for a period of 24 hours resulted in down-regulation of MTP1 expression in the spleen. These data indicate that LPS mediated down-regulation of MTP1 requires a functional TLR4, but that there are non-TLR4 dependent mechanisms for the down-regulation of MTP1 by inflammatory stimuli. In vitro treatment of mouse adherent splenocytes with 5 ug ml of LPS also resulted in down-regulation of MTP1 mRNA. This in vitro down-regulation was not abrogated by co-treatment of cells with pyrrolidinedithiocarbamate (PDTC), a well-characterized inhibitor of NF-KB activation or anti-tumor necrosis factor-a antibodies. In addition, in vitro treatment of mouse splenocytes with recombinant TNF- did not result in down-regulation of MTP1 mRNA. The lack of antagonism between LPS and PDTC and the lack of an effect of TNF- in vitro indicates that NF-B activation may not be required for MTP1 mRNA down-regulation. This inflammation-mediated down-regulation of MTP1 expression in the RES may be a component responsible for iron sequestration in the RES in both acute and chronic inflammatory states.