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BACKGROUND AND PURPOSE: The innate and adaptive immune systems both play important roles in drug‐induced liver injury (DILI). However, the crosstalk between the innate and adaptive immunity in DILI is largely unknown. Extensive crosstalk is likely mandated by co‐stimulatory interactions between these immune systems. OX40 is a co‐stimulatory molecule, but whether it regulates the intrahepatic immune response in DILI remains unknown. EXPERIMENTAL APPROACH: Acute liver injury was induced by paracetamol (acetaminophen), carbon tetrachloride (CCl(4)), and d‐galactosamine/LPS (GalN/LPS) in wild‐type (WT) and Ox40 knockout (KO) mice, and disease progress was compared. KEY RESULTS: Plasma OX40 levels were significantly increased and were augmented in intrahepatic CD4(+) T cells after paracetamol, CCl(4), or GalN/LPS administration. Liver injury in Ox40‐deficient mice was attenuated compared with that in WT mice. Compared with WT mice, hepatic infiltration of Th1 and Th17 cells and macrophages in Ox40 KO mice was reduced. Furthermore, adoptive transfer of Ox40 KO‐CD4(+) T cells to Rag1 (−/−) mice resulted in alleviated liver injury compared with WT‐CD4(+) T‐cell transfer, with reduced liver infiltration of macrophages and pro‐inflammatory cytokine secretion. Moreover, OX40/Fc stimulation in vitro revealed that soluble OX40 enhanced the biological function of murine macrophages, including up‐regulation of genes associated with inflammation and tissue infiltration. Finally, soluble OX40 levels were significantly elevated in DILI patients compared with healthy controls. CONCLUSION AND IMPLICATIONS: OX40 is a key molecule that promotes both pro‐inflammatory macrophage and CD4(+) T‐cell function, exacerbating paracetamol‐induced liver injury. OX40 could serve as a diagnostic index and therapeutic target of DILI.

Background and purpose Fenretinide, a synthetic retinoid derivative first investigated for cancer prevention and treatment, has been shown to ameliorate glucose tolerance, improve plasma lipid profile and reduce body fat mass. These effects, together with its ability to inhibit ceramide synthesis, suggest that fenretinide may have an anti-atherosclerotic action. Experimental approach To this aim, nine-week-old apoE-knockout (EKO) female mice were fed for twelve weeks a Western diet, without (control) or with (0.1% w/w) fenretinide. As a reference, wild-type (WT) mice were treated similarly. Growth and metabolic parameters were monitored throughout the study. Atherosclerosis development was evaluated in the aorta and at the aortic sinus. Blood and lymphoid organs were further characterized with thorough cytological/histological and immunocytofluorimetric analyses. Key results Fenretinide treatment significantly lowered body weight, glucose levels and plasma levels of total cholesterol, triglycerides, and phospholipids. In the liver, fenretinide remarkably reduced hepatic glycogenosis and steatosis driven by the Western diet. Treated spleens were abnormally enlarged, with severe follicular atrophy and massive extramedullary haematopoiesis. Severe renal hemosiderin deposition was observed in treated EKO mice. Treatment resulted in a threefold increase of total leukocytes (WT and EKO) and raised the activated/resting monocyte ratio in EKO mice. Finally, atherosclerosis development was markedly increased at the aortic arch, thoracic and abdominal aorta of fenretinide-treated mice. Conclusions and implications We provide the first evidence that, despite beneficial metabolic effects, fenretinide treatment may enhance the development of atherosclerosis.

Background and purpose Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis, is closely related to metabolic diseases such as obesity and diabetes. Despite an accumulating number of studies, no pharmacotherapy that targets NAFLD has received general approval for clinical use. Experimental approach Inhibition of the sodium-glucose cotransporter 2 (SGLT2) is a promising approach to treat diabetes, obesity, and associated metabolic disorders. In this study, we investigated the effect of a novel SGLT2 inhibitor, NGI001, on NAFLD and obesity-associated metabolic symptoms in high-fat diet (HFD)-induced obese mice. Key results Delayed intervention with NGI001 protected against body weight gain, hyperglycaemia, hyperlipidaemia, and hyperinsulinaemia, compared with HFD alone. Adipocyte hypertrophy was prevented by administering NGI001. NGI001 inhibited impaired glucose metabolism and regulated the secretion of adipokines associated with insulin resistance. In addition, NGI001 supplementation suppressed hepatic lipid accumulation and inflammation but had little effect on kidney function. In-depth investigations showed that NGI001 ameliorated fat deposition and increased AMPK phosphorylation, resulting in phosphorylation of its major downstream target, acetyl-CoA carboxylase, in human hepatocyte HuS-E/2 cells. This cascade ultimately led to the down-regulation of downstream fatty acid synthesis-related molecules and the up-regulation of downstream β oxidation-associated molecules. Surprisingly, NGI001 decreased gene and protein expression of SGLT1 and SGLT2 and glucose uptake in oleic acid-treated HuS-E/2 cells. Conclusion and implications Our findings suggest the novel SGLT2 inhibitor, NGI001 has therapeutic potential to attenuate or delay the onset of diet-induced metabolic diseases and NAFLD.

Background and purpose Hepatic mitochondrial pyruvate carrier (MPC) transports pyruvate into mitochondria. This study investigated the involvement of MPC1 in hepatic glucagon response, in order to identify a possible pharmacological intervention. Experimental approach The correlation between hepatic glucagon response and MPC1 induction was investigated in fasted mice and primary hepatocytes. The effects of ginsenoside Rb1 on MPC1 function were observed. Key results Glucagon challenge raised blood glucose with hepatic MPC1 induction, and inhibition of MPC induction coincided with a reduced rise in blood glucose. cAMP-responsive element-binding protein (CREB) knockdown blocked glucagon-induced MPC1 expression, while CREB overexpression increased MPC1 expression. Luciferase reporter, chromatin immunoprecipitation assay, and promoter mutation confirmed that CREB increased MPC1 transcription through gene promoter induction. CREB regulated transcription co-activator 2 nuclear translocation was also required for CREB to promote MPC1 induction. Glucagon shifted mitochondrial pyruvate towards carboxylation for gluconeogenesis via the opposite regulation of pyruvate dehydrogenase and carboxylase with respect to MPC1 induction. MPC1 induction was necessary for glucagon to promote pyruvate-driven hepatic glucose production (HGP), but glucagon failed to influence HGP from other gluconeogenic substrates routed into the tricarboxylic acid cycle, independent of MPC. Rb1 blocked cAMP signalling by inhibiting AC activity and deactivated CREB by dephosphorylation, possibly contributing to inhibiting MPC1 induction to reduce HGP. Conclusions and implications CREB transcriptionally up-regulates MPC1 to provide pyruvate for gluconeogenesis. Rb1 reduced cAMP formation which consequently reduced CREB-mediated MPC1 induction and thereby might contribute to limiting pyruvate-dependent HGP. These results suggest a therapeutic strategy to reduce hyperglycaemia in diabetes.

Background and purpose Hepatic steatosis may be associated with an increased γ-glutamyltransferase (γ-GT) levels. Ischaemia-reoxygenation (IR) injury causes several deleterious effects. We evaluated the protective effects of a selective inhibitor of γ-GT in experimentally induced IR injury in rats with obesity and steatosis. Experimental approach Otsuka Long-Evans Tokushima Fatty (OLETF) rats with hepatic steatosis were used in the current study. The portal vein and hepatic artery of left lateral and median lobes were clamped to induce ischaemia. Before clamping, 1 ml of saline (IR group) or 1-ml saline containing 1 mg·kg-1 body weight of GGsTop (γ-GT inhibitor; IR-GGsTop group) was injected into the liver via the inferior vena cava. Blood flow was restored after at 30 min of the start of ischaemia. Blood was collected before, at 30 min after ischaemia and at 2 h and 6 h after reoxygenation. All the animals were killed at 6 h and the livers were collected. Key results Treatment with GGsTop resulted in significant reduction of serum ALT, AST and γ-GT levels and hepatic γ-GT, malondialdehyde, 4-hydroxy-2-nonenal and HMGB1 at 6 h after reoxygenation. Inhibition of γ-GT retained normal hepatic glutathione levels. There was prominent hepatic necrosis in IR group, which is significantly reduced in IR-GGsTop group. Conclusion and implications Treatment with GGsTop significantly increased hepatic glutathione content, reduced hepatic MDA, 4-HNE and HMGB1 levels and, remarkably, ameliorated hepatic necrosis after ischaemia-reoxygenation. The results indicated that GGsTop could be an appropriate therapeutic agent to reduce IR-induced liver injury in obesity and steatosis.

Background and purpose The mineralocorticoid receptor (MR) contributes to fibrosis in various tissues, and MR antagonists, like eplerenone, are used to prevent fibrosis. The role of MR antagonists in hepatic fibrosis and cirrhosis is unknown. Here, we investigated the role of MRs and eplerenone in cirrhosis development. Experimental approach Liver fibrosis (5 weeks) and cirrhosis, without (8 weeks) and with ascites (12 weeks), were induced by CCl4 in rats and comprehensively analysed. The effect of eplerenone on the development of cirrhosis with ascites was assessed. MR expression, cellular and subcellular distribution and impact of hypoxia were investigated in vivo and ex vivo. Primary rat hepatocytes and cell lines were used to investigate MR trafficking and transcriptional activity mechanistically. Key results In cirrhosis with ascites, MR mRNA and protein expressions were reduced in hepatocytes of hypoxic areas. While in normoxic areas MRs were mainly cytosolic, the remaining MRs in hypoxic areas were mainly localized in the nuclei, indicating activation followed by translocation and degradation. Accordingly, eplerenone treatment prevented nuclear MR translocation and the worsening of cirrhosis. Exposing hepatocytes ex vivo to hypoxia induced nuclear MR translocation and enhanced transcriptional MR activity at response elements of the NF-κB pathway. Conclusions and implications We showed for the first time that hypoxia leads to a pathogenetic ligand-independent activation of hepatic MRs during cirrhosis resulting in their nuclear translocation and transcriptional activation of the NF-κB pathway. Treatment with eplerenone prevented the worsening of cirrhosis by blocking this ligand-independent activation of the MR.

Background and purpose Hepatic fatty acid metabolism disorder, a key pathogenic mechanism underlying non-alcoholic fatty liver disease (NAFLD), is associated with the hyperacetylation of mitochondrial enzymes. Acyl-CoA synthetase family member 3 (ACSF3), which is involved in the regulation of fatty acid metabolism, was predicted to contain lysine acetylation sites related to the mitochondrial deacetylase sirtuin 3 (SIRT3). The purpose of this study was to explore the underlying mechanism by which SIRT3 deacetylates ACSF3 in NAFLD and the protective effect of the natural phenolic compound protocatechuic acid (PCA) against fatty acid metabolism disorder via the SIRT3/ACSF3 pathway. Experimental approach The role of protocatechuic acid and its molecular mechanism in NAFLD were detected in rats and SIRT3-knockout mice fed a high-fat diet (HFD) and in AML-12 cells treated with palmitic acid (PA). Key results Pharmacological treatment with protocatechuic acid significantly attenuated high-fat diet-induced fatty acid metabolism disorder in NAFLD. Molecular docking assays showed that protocatechuic acid specifically bound SIRT3 as a substrate and increased SIRT3 protein expression. However, the protective role of protocatechuic acid was abolished by SIRT3 knockdown, which increased ACSF3 expression and exacerbated fatty acid metabolism disorder. Mechanistically, SIRT3 was shown to specifically regulate the acetylation and degradation of ACSF3, which govern the capacity of ACSF3 to mediate fatty acid metabolism disorder during NAFLD. Conclusion and implications SIRT3-mediated ACSF3 deacetylation is a novel molecular mechanism in NAFLD therapy and protocatechuic acid confers protection against high-fat diet- and palmitic acid-induced hepatic fatty acid metabolism disorder through the SIRT3/ACSF3 pathway.

BACKGROUND AND PURPOSE: Liver fibrosis is a serious cause of morbidity and mortality worldwide and has no adequate treatment. Accumulating evidence suggests that cannabinoid CB(1) receptors regulate a variety of physiological and pathological processes in the liver, and blockage of CB(1) receptor signalling shows promise as a new therapy for several liver diseases. The aim of this study was to investigate the potential therapeutic effects of CB(1) receptors and a peripheral CB(1) receptor antagonist JD5037 in liver fibrogenesis. EXPERIMENTAL APPROACH: Liver samples from both humans and mouse models were investigated. The peripheral CB(1) receptor antagonist JD5037, β‐arr1 wild type (β‐arr1‐WT) and β‐arr1 knockout (β‐arr1‐KO) littermate models, and primary hepatic stellate cells (HSCs) were also used. The mechanisms underlying CB(1) receptor‐regulated HSCs activation in fibrosis and the therapeutic potential of JD5037 were further analysed. KEY RESULTS: CB(1) receptors were induced in samples from patients with liver fibrosis and from mouse models. These receptors promoted activation of HSCs in liver fibrosis via recruiting β‐arrestin1 and Akt signalling, while blockage of CB(1) receptors with JD5037 attenuated CB(1) receptor‐regulated HSCs activation and liver fibrosis by suppressing β‐arrestin1/Akt signalling. CONCLUSIONS AND IMPLICATIONS: CB(1) receptors promote the activation of HSCs and liver fibrosis via the β‐arrestin1/Akt signalling pathway. The peripheral CB(1) receptor antagonist JD5037 blocked this pathway, the activation of HSCs and liver fibrosis. This compound and the associated pathway may be a novel approach to the treatment of liver fibrosis.

Background and purpose This study investigates the antifibrotic activities and potential mechanisms of costunolide (COS), a natural sesquiterpene compound. Experimental approach Rats subjected to bile duct ligation and mice challenged with CCl4 were used to study the antifibrotic effects of COS in vivo. Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX-2 also served as an in vitro liver fibrosis models. The expression of fibrogenic genes and signaling proteins in the neurogenic locus notch homologue protein 3 (Notch3)-hairy/enhancer of split-1 (HES1) pathway was examined using western blot and/or real-time PCR. Notch3 degradation was analysed using immunofluorescence and coimmunoprecipitation. Key results In animals, COS administration attenuated hepatic histopathological injury and collagen accumulation and reduced the expression of fibrogenic genes. COS time- and dose-dependently suppressed the levels of fibrotic markers in LX-2 cells and mouse pHSCs. Mechanistic studies showed COS destabilized Notch3 and subsequently inhibited the Notch3-HES1 pathway, thus inhibiting HSC activation. Furthermore, COS blocked the WW domain-containing protein 2 (WWP2)/protein phosphatase 1G (PPM1G) interaction and enhanced the effect of WWP2 on Notch3 degradation. Conclusions and implications COS exerted potent antifibrotic effects in vitro and in vivo by disrupting the WWP2/PPM1G complex, promoting Notch3 degradation and inhibiting the Notch3/HES1 pathway. This indicates that COS may be a potential therapeutic candidate for the treatment of liver fibrosis.

BACKGROUND AND PURPOSE: Naringenin, a flavonoid compound with strong anti‐inflammatory activity, attenuated non‐alcoholic fatty liver disease (NAFLD) induced by a methionine‐choline deficient (MCD) diet in mice. However, the mechanisms underlying this suppression of inflammation and NAFLD remain unknown. EXPERIMENTAL APPROACH: WT and NLRP3(−/−) mice were fed with MCD diet for 7 days to induce NAFLD and were given naringenin by gavage at the same time. in vitro experiments used HepG2 cells, primary hepatocytes, and Kupffer cells (KCs) stimulated by LPS or LPS plus oleic acid (OA). KEY RESULTS: Treating WT mice with naringenin (100 mg·kg(−1)·day(−1)) attenuated hepatic lipid accumulation and inflammation in the livers of mice given the MCD diet. NLRP3(−/−) mice showed less hepatic lipid accumulation than WT mice, but naringenin did not ameliorate hepatic lipid accumulation further in NLRP3(−/−) mice. Treating the HepG2 cells with naringenin or NLRP3 inhibitor MCC950 reduced lipid accumulation. Naringenin inhibited activation of the NLRP3/NF‐κB pathway stimulated by OA together with LPS. In KCs isolated from WT mice, naringenin inhibited NLRP3 expression. Naringenin also inhibited lipid deposition, NLRP3 and IL‐1β expression in WT hepatocytes but was not effective in NLRP3(−/−) hepatocytes. After re‐expressing NLRP3 in NLRP3(−/−) hepatocytes by adenovirus, the anti‐lipid deposition effect of naringenin was restored. CONCLUSION AND IMPLICATIONS: Naringenin prevented NAFLD via down‐regulating the NLRP3/NF‐κB signalling pathway both in KCs and in hepatocytes, thus attenuating inflammation in the mice livers.

Background and purpose Ursodeoxycholic acid (UDCA) is the first-line treatment for primary biliary cholangitis, but its effects on the enterohepatic circulation of bile acid (BA) have been under-investigated. Therefore, we studied the influence of UDCA on BA enterohepatic circulation in vivo and the mechanisms by which UDCA affects the BA kinetics. Experimental approach Mice were treated with UDCA and other BAs to observe changes in BA pool and BA transporters involved in enterohepatic circulation. Isotope dilution techniques and biochemical analyses were applied to study BA kinetics after oral administration of UDCA, and the mechanism involved. Key results Oral administration of UDCA in mice reduced the overall BA pool and produced a unique BA profile with high-abundance conjugated UDCA species, including tauroursodeoxycholic acid (TUDCA) and GUDCA. We found increased expression of several main BA transporters in the ileum and liver. BA kinetic experiment showed that feeding UDCA shortened cycling time of BA and accelerated BA enterohepatic circulation. Additionally, we found evidence that the effect of UDCA administration on accelerating BA enterohepatic circulation was due to the inhibition of farnesoid X receptor (FXR) signalling in the ileum and FGF15/19 in the liver. Conclusion and implications Oral administration of UDCA produced a unique BA profile with high-abundance TUDCA and GUDCA and significantly accelerated BA enterohepatic circulation through the inhibition of intestinal FXR signalling and reduced level of FGF15/19, which in turn, induced the expression of BA transporters in the liver. These findings highlight a critical role for UDCA in maintaining the homeostasis of BA enterohepatic circulation in vivo.

Background and purpose Sunitinib is a small-molecule TK inhibitor associated with hepatotoxicity. The mechanisms of its toxicity are still unclear. Experimental approach In the present study, mice were treated with 60, 150, and 450 mg·kg-1 sunitinib to evaluate sunitinib hepatotoxicity. Sunitinib metabolites and endogenous metabolites in liver, serum, faeces, and urine were analysed using ultra-performance LC electrospray ionization quadrupole time-of-flight MS-based metabolomics. Key results Four reactive metabolites and impaired clearance of sunitinib in liver played a dominant role in sunitinib-induced hepatotoxicity. Using a non-targeted metabolomics approach, various metabolic pathways, including mitochondrial fatty acid β-oxidation (β-FAO), bile acids, lipids, amino acids, nucleotides, and tricarboxylic acid cycle intermediates, were disrupted after sunitinib treatment. Conclusions and implications These studies identified significant alterations in mitochondrial β-FAO and bile acid homeostasis. Activation of PPARα and inhibition of xenobiotic metabolism may be of value in attenuating sunitinib hepatotoxicity.

BACKGROUND AND PURPOSE: The molecular mechanism for the pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains elusive. Both farnesoid X receptor (FXR) signalling and endoplasmic reticulum (ER) stress contribute to the progression of NAFLD; however, it is not clear whether the actions of these two pathways are dependent on each other. Moreover, the pharmacological benefits and mechanism of betulinic acid (BA) in controlling metabolic syndrome and NAFLD are largely unknown. EXPERIMENTAL APPROACH: A reporter assay and a time‐resolved FRET assay were used to identify BA as an agonist of the FXR. NAFLD was induced by a methionine and choline‐deficient L‐amino acid diet (MCD) and high‐fat diet (HFD). The pharmacological effects of BA (100 mg·kg(−1)·day(−1)) and potential interactions between hepatic FXR activation and ER stress pathways were evaluated by FXR silencing, Western blot and RT‐PCR analyses using control and FXR(−/−) mice. KEY RESULTS: Activation of the FXR inhibited intracellular PERK/EIF2α/ATF4 and CHOP signalling, thereby alleviating hepatic ER stress, whereas FXR silencing resulted in an opposite effect. Furthermore, we identified BA as an FXR agonist that effectively attenuated the progression of NAFLD and metabolic disorders in both HFD‐ and MCD diet‐fed mice and restored the hepatocellular ER homeostasis by stimulating the FXR signalling pathway and blocking PERK/EIF2α signalling. In contrast, the effects of BA were attenuated in FXR(−/−) mice. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that pharmacological activation of the FXR by BA reduces hepatocellular ER stress and attenuates NAFLD in an animal model of hepatic steatosis.

Background and Purpose The effects of ketamine in attenuating morphine tolerance have been suggested to result from a pharmacodynamic interaction. We studied whether ketamine might increase brain morphine concentrations in acute coadministration, in morphine tolerance and morphine withdrawal. Experimental Approach Morphine minipumps (6mg center dot day(-1)) induced tolerance during 5 days in Sprague-Dawley rats, after which s.c. ketamine (10mg center dot kg(-1)) was administered. Tail flick, hot plate and rotarod tests were used for behavioural testing. Serum levels and whole tissue brain and liver concentrations of morphine, morphine-3-glucuronide, ketamine and norketamine were measured using HPLC-tandem mass spectrometry. Key Results In morphine-naive rats, ketamine caused no antinociception whereas in morphine-tolerant rats there was significant antinociception (57% maximum possible effect in the tail flick test 90min after administration) lasting up to 150min. In the brain of morphine-tolerant ketamine-treated rats, the morphine, ketamine and norketamine concentrations were 2.1-, 1.4- and 3.4-fold, respectively, compared with the rats treated with morphine or ketamine only. In the liver of morphine-tolerant ketamine-treated rats, ketamine concentration was sixfold compared with morphine-naive rats. After a 2 day morphine withdrawal period, smaller but parallel concentration changes were observed. In acute coadministration, ketamine increased the brain morphine concentration by 20%, but no increase in ketamine concentrations or increased antinociception was observed. Conclusions and Implications The ability of ketamine to induce antinociception in rats made tolerant to morphine may also be due to increased brain concentrations of morphine, ketamine and norketamine. The relevance of these findings needs to be assessed in humans.

BACKGROUND AND PURPOSE: Non‐alcoholic steatohepatitis (NASH) is the most severe form of non‐alcoholic fatty liver disease and is a serious public health problem around the world. There are currently no approved treatments for NASH. FGF21 has recently emerged as a promising drug candidate for metabolic diseases. However, the disadvantages of FGF21 as a clinically useful medicine include its short plasma half‐life and poor drug‐like properties. Here, we have explored the effects of PsTag600‐FGF21, an engineered long‐acting FGF21 fusion protein, in mice with NASH and describe some of the underlying mechanisms. EXPERIMENTAL APPROACH: A long‐acting FGF21 was prepared by genetic fusion with a 600 residues polypeptide (PsTag600). We used a choline‐deficient high‐fat diet‐induced model of NASH in mice. The effects on body weight, insulin sensitivity, inflammation and levels of hormones and metabolites were studied first. We further investigated whether PsTag600‐FGF21 attenuated inflammation through the Th17‐IL17A axis and the associated mechanisms. KEY RESULTS: PsTag600‐FGF21 dose‐dependently reduced body weight, blood glucose, and insulin and lipid levels and reversed hepatic steatosis. PsTag600‐FGF21 enhanced fatty acid activation and mitochondrial β‐oxidation in the liver. The profound reduction in hepatic inflammation in NASH mice following PsTag600‐FGF21 was associated with inhibition of IL17A expression in Th17 cells. Furthermore, PsTag600‐FGF21 depended on adiponectin to exert its suppression of Th17 cell differentiation and IL17A expression. CONCLUSIONS AND IMPLICATIONS: Our data have uncovered some of the mechanisms by which PsTag600‐FGF21 suppresses hepatic inflammation and further suggest that PsTag600‐FGF21 could be an effective approach in NASH treatment.

In cor pulmonale, the increased afterload imposed on the right ventricle (RV) generates a maladaptive response, impairing the contractile cardiac function. Oxidative mechanisms play an important role in the pathophysiology and progression of this disease. The administration of pterostilbene (PTS), a phytophenol with antioxidant potential, may represent a therapeutic option. In the present study, we evaluated the effect of PTS complexed with hydroxypropyl-β-cyclodextrin (HPβCD) on hypertrophy, contractile function and oxidative parameters in the RV of rats with pulmonary hypertension, induced by the administration of monocrotaline (MCT).The rats received daily doses of the PTS : HPβCD complex at 25, 50 or 100 mg·kgThe PTS : HPβCD complex reduced the production of NADPH oxidase-dependent superoxide anions and oxidative stress in the RV of MCT-treated rats in a dose-dependent manner. At higher doses it prevented the reduction in FAC and TAPSE in MCT-treated animals.The PTS : HPβCD complex prevented the maladaptative remodelling and protected systolic function in the RV of rats with pulmonary hypertension. These cardioprotective mechanisms may be related, in part, to the antioxidant potential of PTS, favoured by the increased p.o. bioavailability promoted by the presence of HPβCD in the complex.

Background and Purpose Activation of hepatic stellate cells (HSCs) is a crucial step in the pathogenesis of hepatic fibrosis. Histone deacetylase (HDAC) is an attractive target in liver fibrosis because it plays a key role in gene expression and cell differentiation. We have developed a HDAC inhibitor, N-hydroxy-7-(2-naphthylthio)heptanomide (HNHA), and investigated the anti-fibrotic activity of HNHA in vitro and in vivo. Experimental Approach We investigated the anti-fibrotic effect of HNHA on mouse and human HSC activation in vitro and in the liver of bile duct-ligated (BDL) rats in vivo using cell proliferation assays, cell cycle analysis, biochemical assay, immunohistochemistry and Western blots. Liver pathology was assessed with histochemical techniques. Key Results HNHA inhibited proliferation and arrested the cell cycle via p21 induction in HSCs. In addition, HNHA induced apoptosis of HSCs, which was correlated with reduced COX-2 expression, NF-κB activation and cell death signals. HNHA restored liver function and decreased the accumulation of extracellular matrix in the liver via suppression of HSC activation in BDL rats in vivo. HNHA administration also increased survival in BDL rats. Conclusions and Implications HNHA improved liver function, suppressed liver fibrosis and increased survival of BDL rats, accompanied by reduction of cell growth, activation and survival of HSCs. These findings show that HNHA may be a potent anti-fibrosis agent against hepatic fibrosis because of its multi-targeted inhibition of HSC activity in vivo and in vitro.

Background and Purpose The aetiology of inflammation in the liver and vessel wall, leading to non-alcoholic steatohepatitis (NASH) and atherosclerosis, respectively, shares common mechanisms including macrophage infiltration. To treat both disorders simultaneously, it is highly important to tackle the inflammatory status. Exendin-4, a glucagon-like peptide-1 (GLP-1) receptor agonist, reduces hepatic steatosis and has been suggested to reduce atherosclerosis; however, its effects on liver inflammation are underexplored. Here, we tested the hypothesis that exendin-4 reduces inflammation in both the liver and vessel wall, and investigated the common underlying mechanism. Experimental Approach Female APOE 3-Leiden.CETP mice, a model with human-like lipoprotein metabolism, were fed a cholesterol-containing Western-type diet for 5 weeks to induce atherosclerosis and subsequently treated for 4 weeks with exendin-4. Key Results Exendin-4 modestly improved dyslipidaemia, but markedly decreased atherosclerotic lesion severity and area (-33%), accompanied by a reduction in monocyte adhesion to the vessel wall (-42%) and macrophage content in the plaque (-44%). Furthermore, exendin-4 reduced hepatic lipid content and inflammation as well as hepatic CD68+ (-18%) and F4/80+ (-25%) macrophage content. This was accompanied by less monocyte recruitment from the circulation as the Mac-1+ macrophage content was decreased (-36%). Finally, exendin-4 reduced hepatic chemokine expression in vivo and suppressed oxidized low-density lipoprotein accumulation in peritoneal macrophages in vitro, effects dependent on the GLP-1 receptor. Conclusions and Implications Exendin-4 reduces inflammation in both the liver and vessel wall by reducing macrophage recruitment and activation. These data suggest that exendin-4 could be a valuable strategy to treat NASH and atherosclerosis simultaneously. © 2013 The British Pharmacological Society.

β-Caryophyllene (BCP) is a plant-derived FDA approved food additive with anti-inflammatory properties. Some of its beneficial effects in vivo are reported to involve activation of cannabinoid CBIn this study, we investigated the effects of BCP on liver injury induced by chronic plus binge alcohol feeding in mice in vivo by using biochemical assays, real-time PCR and histology analyses. Serum and hepatic BCP levels were also determined by GC/MS.Chronic treatment with BCP alleviated the chronic and binge alcohol-induced liver injury and inflammation by attenuating the pro-inflammatory phenotypic `M1` switch of Kupffer cells and by decreasing the expression of vascular adhesion molecules intercellular adhesion molecule 1, E-Selectin and P-Selectin, as well as the neutrophil infiltration. It also beneficially influenced hepatic metabolic dysregulation (steatosis, protein hyperacetylation and PPAR-α signalling). These protective effects of BCP against alcohol-induced liver injury were attenuated in CBGiven the safety of BCP in humans, this food additive has a high translational potential in treating or preventing hepatic injury associated with oxidative stress, inflammation and steatosis.This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

The in vivo oestrogenicity of genistein and its glycoside genistin is still under debate. The present study aimed to develop a physiologically based kinetic (PBK) model that provides insight in dose-dependent plasma concentrations of genistein aglycone and its metabolites and enables prediction of in vivo oestrogenic effective dose levels of genistein and genistin in humans.A PBK model for genistein and genistin in humans was developed based on in vitro metabolic parameters. The model obtained was used to translate in vitro oestrogenic concentration-response curves of genistein to in vivo oestrogenic dose-response curves for intake of genistein and genistin.The model predicted that genistein-7-O-glucuronide was the major circulating metabolite and that levels of the free aglycone were generally low [0.5-17% of total plasma genistein at oral doses from 0.01 to 50 mg (kg·bw)The present study shows how plasma concentrations of genistein and its metabolites and oestrogenic dose levels of genistein in humans can be predicted by combining in vitro oestrogenicity with PBK model-based reverse dosimetry, eliminating the need for human intervention studies.

Non-alcoholic steatohepatitis (NASH) is characterized by excessive intracellular lipid accumulation, inflammation and hepatic insulin resistance. As the incidence of NASH is increasing worldwide, there is an urgent need to find novel interventional approaches. The pro-inflammatory cytokine IL-1β, generated and released from Kupffer cells, is considered to initiate the development of NASH. AS-1, a synthetic low-molecule mimetic of myeloid differentiation primary response gene 88 (MyD88), disrupts the interaction between the IL-1 receptor and MyD88. Here, we investigated whether AS-1 could attenuate the pathogenesis of NASH with an emphasis on hepatic insulin resistance.Eight-week-old db/db mice were fed a control diet or a methionine- and choline-deficient (MCD) diet. AS-1 (50 mg·kgAS-1 administration significantly ameliorated NASH as evidenced by alanine aminotransferase levels and CD68 levels in livers of MCD-fed mice. AS-1 inhibited the MCD diet-induced activation of caspase 1 and the NLRP3-ASC inflammasome, and also reduced the enhanced levels of ROS, malondialdehyde, 3-nitrotyrosine, NADPH oxidase complex and CYP reductase-associated cytochrome p450 2E1 (CYP2E1) expression in the liver. In addition, AS-1 decreased ROS, inflammasome activation and IL-1β production in free fatty acid-LPS-treated Kupffer cells. Finally, pretreatment with AS-1 significantly ameliorated gluconeogenesis and insulin desensitization induced by IL-1β, probably by blocking the interaction between MyD88 and the IL-1 receptor.Our results indicate that AS-1 can ameliorate NASH and hepatic insulin resistance and could be considered as a potential strategy for the prevention and treatment of NASH.

Background and purpose: There is major evidence for the strong bi-directional interrelation of parenchymal cell apoptosis and leukocyte accumulation and inflammation in acute liver injury. Therefore, the aim of this in vivo study was to investigate the anti-apoptotic and anti-inflammatory potential of antileukoproteinase (ALP) in a murine model of acute liver failure. Experimental approach: C57BL/6J mice were given galactosamine (D-GalN) and E. coli lipopolysaccharide (LPS) followed by administration of saline or ALP. Besides survival rate, hepatic tissue damage and inflammatory response were analyzed by intravital fluorescence microscopy 6 hours after treatment. In addition, immunohistochemical analysis of NFκB-p65 and hepatocellular apoptosis, plasma levels of AST/ALT, TNF-α and IL-10 were determined. Key results: Administration of D-GalN/LPS provoked hepatic damage, including marked leukocyte recruitment and microvascular perfusion failure, as well as hepatocellular apoptosis and enzyme release. NFκB-p65 became increasingly detectable in hepatocellular nuclei, accompanied by a rise of TNF-α and IL-10 plasma levels. ALP markedly reduced intrahepatic leukocyte accumulation, nuclear translocation of NFκB and plasma levels of TNF-α and IL-10. Moreover, liver enzyme levels indicated the absence of necrotic parenchymal cell death. In contrast, ALP failed to block both apoptosis and caspase-3 levels and the mortality rate of ALP-treated animals was comparable to that of saline-treated mice. Conclusions and implications: ALP could effectively prevent D-GalN/LPS-associated intrahepatic inflammatory responses by inhibition of NFκB activity, but not apoptosis-driven mortality. Thus, a protease-inactivating approach such as application of ALP seems to be inadequate in damaged liver where apoptosis represents the predominant mode of cell death. British Journal of Pharmacology (2007) 151, 406–413; doi:10.1038/sj.bjp.0707230

Background and purpose: Activation of the pregnane X receptor (PXR) has been shown to protect against cholestatic hepatotoxicity. As PXR alters the expression of numerous hepatic bile acid transporters, we sought to delineate their potential role in hepatoprotection. Experimental approach: Wild-type (PXR+/+) and PXR-null (PXR-/-) mice were fed a 1% cholic acid (CA) diet with or without the PXR activator, PCN. Liver function was assessed along with the corresponding changes in hepatic gene expression. Key results: CA administration caused significant hepatotoxicity in PXR+/+ mice and was associated with induction of several FXR and PXR regulated genes, which encode for bile acid transport and metabolizing proteins. Compared to CA alone, co-administration of PCN to CA-fed PXR+/+ mice significantly decreased hepatotoxicity and was associated with induction of MRP3 mRNA as well as CYP3A11 mRNA and functional activity. Unexpectedly, PXR-/- mice, which expressed significantly higher basal and CA-induced levels of MRP2, MRP3, OSTα, OSTβ, OATP2 and CYP3A11, were dramatically less sensitive to CA hepatotoxicity than PXR+/+ mice. Conclusions: Protection of PXR+/+ mice against CA-induced hepatotoxicity by PCN is associated with the induction of MRP3 and CYP3A11 expression. Resistance against CA-induced hepatotoxicity in PXR-/- mice may result from higher basal and induced expression of bile acid transporters, particularly MRP3. These findings emphasize the importance of transport by MRP3 and metabolism as major protective pathways against cholestatic liver injury. British Journal of Pharmacology (2007) 151, 367–376; doi:10.1038/sj.bjp.0707235

Autophagy is an essential cytoprotective system that is rapidly activated in response to various stimuli including inflammation and microbial infection. Genipin, an aglycon of geniposide found in gardenia fruit, is well known to have anti-inflammatory, antibacterial and antioxidative properties. This study examined the protective mechanisms of genipin against sepsis, with particular focus on the autophagic signalling pathway.Mice were subjected to sepsis by caecal ligation and puncture (CLP). Genipin (1, 2.5 and 5 mg·kg(-1) ) or vehicle (saline) was injected i.v. immediately (0 h) after CLP, and chloroquine (60 mg·kg(-1) ), an autophagy inhibitor, was injected i.p. 1 h before CLP. Blood and liver tissues were isolated 6 h after CLP.Genipin improved survival rate and decreased serum levels of aminotransferases and pro-inflammatory cytokines after CLP; effects abolished by chloroquine. The liver expression of autophagy-related protein (Atg)12-Atg5 conjugate increased after CLP, and this increase was enhanced by genipin. CLP decreased Atg3 protein liver expression, and genipin attenuated this decrease. CLP impaired autophagic flux, as indicated by increased liver expression of microtubule-associated protein-1 light chain 3-II and sequestosome-1/p62 protein; this impaired autophagic flux was restored by genipin, and chloroquine abolished this effect. Genipin also attenuated the decreased expression of lysosome-associated membrane protein-2 and Rab7 protein and increased expression of calpain 1 protein induced by CLP in the liver.Our findings suggest that genipin protects against septic injury by restoring impaired autophagic flux. Therefore, genipin might be a potential therapeutic agent for the treatment of sepsis.

Small-molecule inhibitors of prolyl hydroxylase (PHD) enzymes are a novel target for the treatment of anaemia and functional iron deficiency (FID). Other than being orally bioavailable, the differentiation of PHD inhibitors from recombinant human erythropoietin (rhEPO) has not been demonstrated.JNJ-42905343 was identified and characterized as a novel inhibitor of PHD and its action was compared with rhEPO in healthy rats and in a rat model of inflammation-induced anaemia and FID [peptidoglycan-polysaccharide (PGPS) model].Oral administration of JNJ-42905343 to healthy rats increased the gene expression of cytochrome b (DcytB) and divalent metal-ion transporter 1 (DMT1) in the duodenum, and increased plasma EPO. Repeated administration of JNJ-42905343 for 28 days increased blood haemoglobin, mean corpuscular haemoglobin (MCH) and mean corpuscular volume (MCV). The serum iron concentration was increased with low doses (0.3 mg·kg(-1) ) but reduced at high doses (6 mg·kg(-1) ). In PGPS-treated rats, administration of JNJ-42905343 for 28 days corrected FID and anaemia, as reflected by increased blood haemoglobin, MCH and MCV. Increased expression of DcytB and DMT1 genes in the duodenum resulting in increased iron availability was defined as the mechanism for these effects. rhEPO did not affect DcytB and DMT1 and was not effective in PGPS-treated rats.PHD inhibition has a beneficial effect on iron metabolism in addition to stimulating the release of EPO. Small-molecule inhibitors of PHD such as JNJ-42905343 represent a mechanism distinct from rhEPO to treat anaemia and FID.

Salsalate (salicylsalicylic acid) is an anti-inflammatory drug that was recently found to exert beneficial metabolic effects on glucose and lipid metabolism. Although its utility in the prevention and management of a wide range of vascular disorders, including type 2 diabetes and metabolic syndrome has been suggested before, the potential of salsalate to protect against non-alcoholic steatohepatitis (NASH) remains unclear. The aim of the present study was therefore to ascertain the effects of salsalate on the development of NASH.Transgenic APOE*3Leiden.CETP mice were fed a high-fat and high-cholesterol diet with or without salsalate for 12 and 20 weeks. The effects on body weight, plasma biochemical variables, liver histology and hepatic gene expression were assessed.Salsalate prevented weight gain, improved dyslipidemia and insulin resistance and ameliorated diet-induced NASH, as shown by decreased hepatic microvesicular and macrovesicular steatosis, reduced hepatic inflammation and reduced development of fibrosis. Salsalate affected lipid metabolism by increasing β-oxidation and decreasing lipogenesis, as shown by the activation of PPAR-α, PPAR-γ co-activator 1β, RXR-α and inhibition of genes controlled by the transcription factor MLXIPL/ChREBP. Inflammation was reduced by down-regulation of the NF-κB pathway, and fibrosis development was prevented by down-regulation of TGF-β signalling.Salsalate exerted a preventive effect on the development of NASH and progression to fibrosis. These data suggest a clinical application of salsalate in preventing NASH.

Recent reports have suggested that salidroside could protect cardiomyocytes from oxidative injury and stimulate glucose uptake in skeletal muscle cells by activating AMP-activated protein kinase (AMPK). The aim of this study was to evaluate the therapeutic effects of salidroside on diabetic mice and to explore the underlying mechanisms.The therapeutic effects of salidroside on type 2 diabetes were investigated. Increasing doses of salidroside (25, 50 and 100 mg·kg(-1) ·day(-1)) were administered p.o. to db/db mice for 8 weeks. Biochemical analysis and histopathological examinations were conducted to evaluate the therapeutic effects of salidroside. Primary cultured mouse hepatocytes were used to further explore the underlying mechanisms in vitro.Salidroside dramatically reduced blood glucose and serum insulin levels and alleviated insulin resistance. Hypolipidaemic effects and amelioration of liver steatosis were observed after salidroside administration. In vitro, salidroside dose-dependently induced an increase in the phosphorylations of AMPK and PI3K/Akt, as well as glycogen synthase kinase 3β (GSK3β) in hepatocytes. Furthermore, salidroside-stimulated AMPK activation was found to suppress the expression of PEPCK and glucose-6-phosphatase. Salidroside-induced AMPK activation also resulted in phosphorylation of acetyl CoA carboxylase, which can reduce lipid accumulation in peripheral tissues. In isolated mitochondria, salidroside inhibited respiratory chain complex I and disturbed oxidation/phosphorylation coupling and moderately depolarized the mitochondrial membrane potential, resulting in a transient increase in the AMP/ATP ratio.Salidroside exerts an antidiabetic effect by improving the cellular metabolic flux through the activation of a mitochondria-related AMPK/PI3K/Akt/GSK3β pathway.

The clinical effects of anti-angiogenic agents remain controversial. Therefore, elucidating the pharmacological properties of these compounds is a pivotal issue.The effects of treatment with sunitinib on tumour and normal tissues of mice bearing C-26 adenocarcinoma cells were analysed by matrix-assisted laser desorption ionization MS imaging (MALDI-MSI). Expression of the key targets of sunitinib--angiogenic receptors--was studied by immunofluorescent labelling.MALDI-MS assays showed that sunitinib and its fragment ions were present throughout tumour and normal tissues. Major metabolites were identified in blood and solid tissues, while minor drug metabolites were detectable only in blood. Tumour growth and intratumour VEGF receptor-2 expressions were significantly reduced in sunitinib-treated mice, while the expression of the other targeted receptors, PDGF receptor -α or -β and fibroblast growth factor receptor-1, remained unaffected. Within tumour tissue, the close proximity of sunitinib metabolites to the precursor ion suggested in situ metabolism of the administered drug. There were intratumour areas where the signal intensity of sunitinib correlated with expression of VEGF receptor-2.This is the first study that demonstrates MALDI-MSI is a versatile platform to study the intratumour localization of an unlabelled anti-angiogenic drug. The combination of MALDI-MSI and immunofluorescence analysis can provide further insights into the molecular interaction of drug compounds and their targets within tumour tissue.

The haematopoietic activity of erythropoietin (EPO) is mediated by the classic EPO receptor (EpoR) homodimer, whereas tissue-protective effects are mediated by a heterocomplex between EpoR and the β-common receptor (βcR). Here, we investigated the effects of a novel, selective ligand of this heterocomplex - pyroglutamate helix B surface peptide (pHBSP) - in mice fed a diet enriched in sugars and saturated fats.Male C57BL/6J mice were fed a high-fat high-sucrose diet (HFHS) for 22 weeks. pHBSP (30 μg·kg(-1) s.c.) was administered for the last 11 weeks. Biochemical assays, histopathological and immunohistochemical examinations and Western blotting were performed on serum and target organs (liver, kidney and skeletal muscle).Mice fed with HFHS diet exhibited insulin resistance, hyperlipidaemia, hepatic lipid accumulation and kidney dysfunction. In gastrocnemius muscle, HFHS impaired the insulin signalling pathway and reduced membrane translocation of glucose transporter type 4 and glycogen content. Treatment with pHBSP ameliorated renal function, reduced hepatic lipid deposition, and normalized serum glucose and lipid profiles. These effects were associated with an improvement in insulin sensitivity and glucose uptake in skeletal muscle. Diet-induced overproduction of the myokines IL-6 and fibroblast growth factor-21 were attenuated by pHBSP and, most importantly, pHBSP markedly enhanced mitochondrial biogenesis in skeletal muscle.Chronic treatment of mice with an EPO derivative, devoid of haematopoietic effects, improved metabolic abnormalities induced by a high-fat high-sucrose diet, by affecting several levels of the insulin signalling and inflammatory cascades within skeletal muscle, while enhancing mitochondrial biogenesis.

The pharmacological properties of particular receptors have recently been suggested to vary under different conditions. We compared the pharmacological properties of the α1B -adrenoceptor subtype in various tissue preparations and under various conditions.[(3) H]-prazosin binding to α1B -adrenoceptors in rat liver (segments, dispersed hepatocytes and homogenates) was assessed and the pharmacological profiles were compared with the functional and binding profiles in rat carotid artery and recombinant α1B -adrenoceptors.In association and saturation-binding experiments with rat liver, binding affinity for [(3) H]-prazosin varied significantly between preparations (KD value approximately ten times higher in segments than in homogenates). The binding profile for various drugs in liver segments also deviated from the representative α1B -adrenoceptor profile observed in liver homogenates and recombinant receptors. L-765,314 and ALS-77, selective antagonists of α1B -adrenoceptors, showed high binding and antagonist affinities in liver homogenates and recombinant α1B -adrenoceptors. However, binding affinities for both ligands in the segments of rat liver and carotid artery were 10 times lower, and the antagonist potencies in α1B -adrenoceptor-mediated contractions of carotid artery were more than 100 times lower than the representative α1B -adrenoceptor profile.In contrast to the consistent profile of recombinant α1B -adrenoceptors, the pharmacological profile of native α1B -adrenoceptors of rat liver and carotid artery varied markedly under various receptor environments, showing significantly different binding properties between intact tissues and homogenates, and dissociation between functional and binding affinities. In addition to conventional 'subtype' characterization, 'phenotype' pharmacology must be considered in native receptor evaluations in vivo and in future pharmacotherapy.

Erythropoietin (EPO) is used to treat anaemia associated with chronic kidney disease (CKD). Hypoxia is associated with anaemia and is known to cause a decrease in cytochrome P450 (P450) expression. As EPO production is regulated by hypoxia, we investigated the role of EPO on P450 expression and function.Male Wistar rats were subjected to a 0.7% adenine diet for 4 weeks to induce CKD. The diet continued for an additional 2 weeks while rats received EPO by i.p. injection every other day. Following euthanasia, hepatic P450 mRNA and protein expression were determined. Hepatic enzyme activity of selected P450s was determined and chromatin immunoprecipitation was used to characterize binding of nuclear receptors involved in the transcriptional regulation of CYP2C and CYP3A.EPO administration decreased hepatic mRNA and protein expression of CYP3A2 (P0.05), but not CYP2C11. Similarly, EPO administration decreased CYP3A2 protein expression by 81% (P0.001). A 32% decrease (P0.05) in hepatic CYP3A enzymatic activity (Vmax ) was observed for the formation of 6βOH-testosterone in the EPO-treated group. Decreases in RNA pol II recruitment (P0.01), hepatocyte nuclear factor 4α binding (P0.05) and pregnane X receptor binding (P0.01) to the promoter region of CYP3A were also observed in EPO-treated rats.Our data show that EPO decreases the expression and function of CYP3A, but not CYP2C in rat liver.

Doxorubicin-based chemotherapy induces cardiotoxicity, which limits its clinical application. We previously reported the protective effects of quercetin against doxorubicin-induced hepatotoxicity. In this study, we tested the effects of quercetin on the expression of Bmi-1, a protein regulating mitochondrial function and ROS generation, as a mechanism underlying quercetin-mediated protection against doxorubicin-induced cardiotoxicity.Effects of quercetin on doxorubicin-induced cardiotoxicity was evaluated using H9c2 cardiomyocytes and C57BL/6 mice. Changes in apoptosis, mitochondrial function, oxidative stress and related signalling were evaluated in H9c2 cells. Cardiac function, serum enzyme activity and reactive oxygen species (ROS) generation were measured in mice after a single injection of doxorubicin with or without quercetin pre-treatment.In H9c2 cells, quercetin reduced doxorubicin-induced apoptosis, mitochondrial dysfunction, ROS generation and DNA double-strand breaks. The quercetin-mediated protection against doxorubicin toxicity was characterized by decreased expression of Bid, p53 and oxidase (p47 and Nox1) and by increased expression of Bcl-2 and Bmi-1. Bmi-1 siRNA abolished the protective effect of quercetin against doxorubicin-induced toxicity in H9c2 cells. Furthermore, quercetin protected mice from doxorubicin-induced cardiac dysfunction that was accompanied by reduced ROS levels and lipid peroxidation, but enhanced the expression of Bmi-1 and anti-oxidative superoxide dismutase.Our results demonstrate that quercetin decreased doxorubicin-induced cardiotoxicity in vitro and in vivo by reducing oxidative stress by up-regulation of Bmi-1 expression. The findings presented in this study have potential applications in preventing doxorubicin-induced cardiomyopathy.

CYP2D6 metabolizes many centrally acting drugs, neurotoxins and endogenous neurochemicals, and differences in brain levels of CYP2D have been associated with brain function and drug response. Alcohol consumers and smokers have higher levels of CYP2D6 in brain, but not liver, suggesting ethanol and/or nicotine may induce human brain CYP2D6. We investigated the independent and combined effects of chronic ethanol self-administration and nicotine treatment on CYP2D expression in African green monkeys.Forty monkeys were randomized into control, ethanol-only, nicotine-only and ethanol + nicotine groups. Two groups voluntarily self-administered 10% ethanol in sucrose solution for 4 h·day(-1) , whereas two groups consumed sucrose solution on the same schedule. Two groups received daily s.c. injections of 0.5 mg·kg(-1) nicotine in saline bid, whereas two groups were injected with saline on the same schedule.Both nicotine and ethanol dose-dependently increased CYP2D in brain; brain mRNA was unaffected, and neither drug altered hepatic CYP2D protein or mRNA. The combination of ethanol and nicotine increased brain CYP2D protein levels to a greater extent than either drug alone (1.2-2.2-fold, P0.05 among the eight brain regions assessed). Immunohistochemistry revealed the induction of brain CYP2D protein within specific cell types and regions in the treatment groups.Ethanol and nicotine increase brain CYP2D protein levels in monkeys, in a region and treatment-specific manner, suggesting that CNS drug responses, neurodegeneration and personality may be affected among people who consume alcohol and/or nicotine.

Aleglitazar is a dual PPARα/γ agonist but little is known about its effects on vascular function and atherogenesis. Hence, we characterized its effects on circulating angiogenic cells (CAC), neoangiogenesis, endothelial function, arteriogenesis and atherosclerosis in mice.C57Bl/6 wild-type (WT, normal chow), endothelial NOS (eNOS)(-/-) (normal chow) and ApoE(-/-) (Western-type diet) mice were treated with aleglitazar (10 mg·kg(-1) ·day(-1) , i.p.) or vehicle.Aleglitazar enhanced expression of PPARα and PPARγ target genes, normalized glucose tolerance and potently reduced hepatic fat in ApoE(-/-) mice. In WT mice, but not in eNOS(-/-) , aleglitazar up-regulated Sca-1/VEGFR2-positive CAC in the blood and bone marrow and up-regulated diLDL/lectin-positive CAC. Aleglitazar augmented CAC migration and enhanced neoangiogenesis. In ApoE(-/-) mice, aleglitazar up-regulated CAC number and function, reduced markers of vascular inflammation and potently improved perfusion restoration after hindlimb ischaemia and aortic endothelium-dependent vasodilatation. This was associated with markedly reduced formation of atherosclerotic plaques. In human cultured CAC from healthy donors and patients with coronary artery disease with or without diabetes mellitus, aleglitazar increased migration and colony-forming units in a concentration-dependent manner. Furthermore, oxidative stress-induced CAC apoptosis and expression of p53 were reduced, while telomerase activity and expression of phospho-eNOS and phospho-Akt were elevated. Comparative agonist and inhibitor experiments revealed that aleglitazar's effects on CAC migration and colony-forming units were mediated by both PPARα and PPARγ signalling and required Akt.Aleglitazar augments the number, function and survival of CAC, which correlates with improved vascular function, enhanced arteriogenesis and prevention of atherosclerosis in mice.

Naturally occurring splice variants of human CAR (hCAR), including hCAR-SV23 (insertion of amino acids SPTV) and hCAR-SV24 (APYLT), have been shown to be expressed in liver. However, little is known regarding how hCAR-SV23 and hCAR-SV24 are activated. Therefore, we investigated the mode of activation of these hCAR splice variants.Cell-based reporter gene assays, including ligand-binding domain transactivation assays and coactivator recruitment assays, were conducted on cultured HepG2 cells transfected with various constructs and treated with 3-hydroxyflavone or a hydroxylated (galangin, datiscetin, kaempferol, morin, quercetin or myricetin) or methylated (isorhamnetin, tamarixetin, or syringetin) analogue.Among the flavonols investigated, only 3-hydroxyflavone increased hCAR-SV23 and hCAR-SV24 activities. 3-Hydroxyflavone did not transactivate the ligand-binding domain of these isoforms or recruit steroid receptor coactivators (SRC-1, SRC-2, or SRC-3). By comparison, 3-hydroxyflavone, galangin, datiscetin, kaempferol, quercetin, isorhamnetin and tamarixetin activated hCAR-WT, whereas none of the flavonols activated hCAR-SV25 (both SPTV and APYLT insertions). The flavonols 3-Hydroxyflavone, galangin, quercetin and tamarixetin transactivated the ligand-binding domain of hCAR-WT, but only 3-hydroxyflavone recruited SRC-1, SRC-2 and SRC-3 to the receptor.hCAR-SV23 and hCAR-SV24 can be activated by a mechanism that does not involve the ligand-binding domain of the receptor or recruitment of SRC-1, SRC-2, or SRC-3. 3-Hydroxyflavone and its structural analogues activated hCAR in an isoform-selective and chemical-specific manner. Overall, our study provides insight into a novel mode of ligand activation of hCAR-SV23 and hCAR-SV24.

Background and Purpose The dual role of the constitutive androstane receptor (CAR) as both a xenosensor and a regulator of endogenous energy metabolism (lipogenesis and gluconeogenesis) has recently gained acceptance. Here, we investigated the effects of 4-[(4R,6R)-4,6-diphenyl-1,3-dioxan-2-yl]-N,N-dimethylaniline (transpDMA), an effective CAR activator, on the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in rat livers. Experimental Approach The effects of transpDMA were investigated in normal and high-fat diet-fed Wistar rats using real-time PCR, Western blotting, chromatin immunoprecipitation assays (ChIP), glucose tolerance test and insulin tolerance test. Key Results The expression of the gluconeogenic enzymes PEPCK and G6Pase was repressed by transpDMA treatment under fasting conditions. Long-term CAR activation by transpDMA significantly reduced fasting blood glucose levels and improved glucose homeostasis and insulin sensitivity in high-fat diet-fed rats. The metabolic benefits of CAR activation by transpDMA may have resulted from the inhibition of hepatic gluconeogenic genes. ChIP assays demonstrated that transpDMA prevented the binding of forkhead box O1 (FOXO1) to insulin response sequences in the PEPCK and G6Pase gene promoters in rat livers. Moreover, transpDMA-activated CAR inhibited hepatocyte nuclear factor-4α (HNF4α) transactivation by competing with HNF4α for binding to the specific binding element (DR1-site) in the gluconeogenic gene promoters. Conclusions and Implications Our results provide evidence to support the conclusion that transpDMA inhibits the gluconeogenic genes PEPCK and G6Pase through suppression of HNF4α and FOXO1 transcriptional activity.

We have previously shown that isoprenaline-induced cardiac hypertrophy causes significant changes in the expression of cytochromes P450 (CYP) and soluble epoxide hydrolase (sEH) genes. Therefore, it is important to examine whether the inhibition of sEH by 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) will protect against isoprenaline-induced cardiac hypertrophy.Male Sprague-Dawley rats were treated with TUPS (0.65 mg kg(-1) day(-1), p.o.), isoprenaline (5 mg kg(-1) day(-1), i.p.) or the combination of both. In vitro H9c2 cells were treated with isoprenaline (100 μM) in the presence and absence of either TUPS (1 μM) or 11,12 EET (1 μM). The expression of hypertrophic, fibrotic markers and different CYP genes were determined by real-time PCR.Isoprenaline significantly induced the hypertrophic, fibrotic markers as well as the heart to body weight ratio, which was significantly reversed by TUPS. Isoprenaline also caused an induction of CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP4A3 and CYP4F4 gene expression and TUPS significantly inhibited this isoprenaline-mediated effect. Moreover, isoprenaline significantly reduced 5,6-, 8,9-, 11,12- and 14,15-EET and increased their corresponding 8,9-, 11,12- and 14,15-dihydroxyeicosatrienoic acid (DHET) and the 20-HETE metabolites. TUPS abolished these isoprenaline-mediated changes in arachidonic acid (AA) metabolites. In H9c2 cells, isoprenaline caused a significant induction of ANP, BNP and EPHX2 mRNA levels. Both TUPS and 11,12-EET significantly decreased this isoprenaline-mediated induction of ANP, BNP and EPHX2.TUPS partially protects against isoprenaline-induced cardiac hypertrophy, which confirms the role of sEH and CYP enzymes in the development of cardiac hypertrophy.

The amelioration of insulin resistance by bilobetin is closely related to its hypolipidaemic effect. The aim of the present study was to determine the insulin-sensitizing mechanism of bilobetin by elucidating its effect on lipid metabolism.Rats fed a high-fat diet were treated with bilobetin for either 4 or 14 days before applying a hyperinsulinaemic-euglycaemic clamp. Triglyceride and fatty acids labelled with radioactive isotopes were used to track the transportation and the fate of lipids in tissues. The activity of lipid metabolism-related enzymes and β-oxidation rate were measured. Western blot was used to investigate the phosphorylation, translocation and expression of PPARα in several tissues and cultured cells. The location of amino acid residues subjected to phosphorylation in PPARα was also studied.Bilobetin ameliorated insulin resistance, increased the hepatic uptake and oxidation of lipids, reduced very-low-density lipoprotein triglyceride secretion and blood triglyceride levels, enhanced the expression and activity of enzymes involved in β-oxidation and attenuated the accumulation of triglycerides and their metabolites in tissues. Bilobetin also increased the phosphorylation, nuclear translocation and activity of PPARα accompanied by elevated cAMP level and PKA activity. Threonine-129-alanine and/or serine-163-alanine mutations on the PPARα genes and PKA inhibitors prevented the effects of bilobetin on PPARα. However, cells overexpressing PKA appeared to stimulate the phosphorylation, nuclear translocation and activity of PPARα.Bilobetin treatment ameliorates hyperlipidaemia, lipotoxicity and insulin resistance in rats by stimulating PPARα-mediated lipid catabolism. PKA activation is crucial for this process.

Hydrogen sulphide (H(2) S) is gaining acceptance as a gaseous signal molecule. However, mechanisms regarding signal termination are not understood. We used stigmatellin and antimycin A, inhibitors of sulphide quinone reductase (SQR), to test the hypothesis that the catabolism of H(2) S involves SQR.H(2) S production and consumption were determined in living and intact mouse brain, liver and colonic muscularis externa using gas chromatography and HPLC. Expressions of SQR, ethylmalonic encephalopathy 1 (Ethe1) and thiosulphate transferase (TST; rhodanese) were determined by RT-PCR and immunohistochemistry.In the colonic muscularis externa, H(2) (35) S was catabolized to [(35) S]-thiosulphate and [(35) S]-sulphate, and stigmatellin reduced both the consumption of H(2) (35) S and formation of [(35) S]-thiosulphate. Stigmatellin also enhanced H(2) S release by the colonic muscularis externa. In the brain, catabolism of H(2) (35) S to [(35) S]-thiosulphate and [(35) S]-sulphate, which was stigmatellin-insensitive, partially accounted for H(2) (35) S consumption, while the remainder was captured as unidentified (35) S that was probably bound to proteins. Levels of mRNA encoding SQR were higher in the colonic muscularis externa and the liver than in the brain.These data support the concept that termination of endogenous H(2) S signalling in the colonic muscularis externa occurs via catabolism to thiosulphate and sulphate partially via a mechanism involving SQR. In the brain, it appears that H(2) S signal termination occurs partially through protein sequestration and partially through catabolism not involving SQR. As H(2) S has beneficial effects in animal models of human disease, we suggest that selective inhibition of SQR is an attractive target for pharmaceutical development.

Peripheral blockade of cannabinoid CB(1) receptors has been proposed as a safe and effective therapy against obesity, putatively devoid of the adverse psychiatric side effects of centrally acting CB(1) receptor antagonists. In this study we analysed the effects of LH-21, a peripherally acting neutral cannabinoid receptor antagonist with poor brain penetration, in an animal model of diet-induced obesity.To induce obesity, male Wistar rats were fed a high-fat diet (HFD; 60 kcal% fat) whereas controls received a standard diet (SD; 10 kcal% fat). Following 10 weeks of feeding, animals received a daily i.p. injection of vehicle or 3 mg·kg(-1) LH-21 for 10 days. Plasma and liver samples were used for biochemical analyses whereas visceral fat-pad samples were analysed for lipid metabolism gene expression using real-time RT-PCR. In addition, the potential of LH-21 to interact with hepatic cytochrome P450 isoforms and cardiac human Ether-à-go-go Related Gene (hERG) channels was evaluated.LH-21 reduced feeding and body weight gain in HFD-fed animals compared with the control group fed SD. In adipose tissue, this effect was associated with decreased gene expression of: (i) leptin; (ii) lipogenic enzymes, including SCD-1; (iii) CB(1) receptors; and (iv) both PPARα and PPARγ. Although there were no significant differences in plasma parameters between HFD- and SD-fed rats, LH-21 did not seem to induce hepatic, cardiac or renal toxicity.These results support the hypothesis that treatment with the peripherally neutral acting CB(1) receptor antagonist, LH-21, may promote weight loss through modulation of visceral adipose tissue.

Soluble epoxide hydrolase inhibitors (sEHIs) possess anti-inflammatory, antiatherosclerotic, antihypertensive and analgesic properties. The pharmacokinetics (PK) and pharmacodynamics in terms of inhibitory potency of sEHIs were assessed in non-human primates (NHPs). Development of a sEHI for use in NHPs will facilitate investigations on the role of sEH in numerous chronic inflammatory conditions.PK parameters of 11 sEHIs in cynomolgus monkeys were determined after oral dosing with 0.3 mg·kg(-1). Their physical properties and inhibitory potency in hepatic cytosol of cynomolgus monkeys were examined. Dose-dependent effects of the two inhibitors 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) and the related acetyl piperidine derivative, 1-trifluoromethoxyphenyl-3-(1-acetylpiperidin-4-yl) urea (TPAU), on natural blood eicosanoids, were determined.Among the inhibitors tested, TPPU and two 4-(cyclohexyloxy) benzoic acid urea sEHIs displayed high plasma concentrations (10 × IC(50)), when dosed orally at 0.3 mg·kg(-1). Although the 4-(cyclohexyloxy) benzoic acid ureas were more potent against monkey sEH than piperidyl ureas (TPAU and TPPU), the latter compounds showed higher plasma concentrations and more drug-like properties. The C(max) increased with dose from 0.3 to 3 mg·kg(-1) for TPPU and from 0.1 to 3 mg·kg(-1) for TPAU, although it was not linear over this range of doses. As an indication of target engagement, ratios of linoleate epoxides to diols increased with TPPU administration.Our data indicate that TPPU is suitable for investigating sEH biology and the role of epoxide-containing lipids in modulating inflammatory diseases in NHPs.

Background and Purpose: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. Experimental Approach: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [ 35S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. Key Results: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys99 on CXCR1 and the non-conserved residue Asp 293 on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. Conclusion and Implications: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases. Fil: Bertini, R.. Dompé; Italia Fil: Barcelos, L. S.. Universidade Federal de Minas Gerais; Brasil Fil: Beccari, A. R.. Dompé; Italia Fil: Cavalieri, B.. Dompé; Italia. Università di Torino; Italia Fil: Moriconi, A.. Dompé; Italia Fil: Bizzarri, C.. Dompé; Italia Fil: Di Benedetto, P.. Dompé; Italia Fil: Di Giacinto, C.. Dompé; Italia Fil: Gloaguen, I.. Dompé; Italia Fil: Galliera, E.. Università degli Studi di Milano; Italia Fil: Corsi, M. M.. Università degli Studi di Milano; Italia. Policlinico San Donato IRCCS; Italia Fil: Russo, R. C.. Universidade Federal de Minas Gerais; Brasil Fil: Andrade, S. P.. Universidade Federal de Minas Gerais; Brasil Fil: Cesta, M. C.. Dompé; Italia Fil: Nano, G.. Dompé; Italia Fil: Aramini, A.. Dompé; Italia Fil: Cutrin, Juan Carlos. Università di Torino; Italia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Cardiológicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Cardiológicas; Argentina Fil: Locati, M.. Università degli Studi di Milano; Italia Fil: Allegretti, M.. Dompé; Italia Fil: Teixeira, M. M.. Universidade Federal de Minas Gerais; Brasil

Mitochondria are involved in the toxicity of several compounds, retro-control of gene expression and apoptosis activation. The effect of mitochondrial genome (mtDNA) depletion on changes in ABC transporter protein expression in response to bile acids and paracetamol was investigated.Hepa 1-6 mouse hepatoma cells with 70% decrease in 16S/18S rRNA ratio (Rho cells) were obtained by long-term treatment with ethidium bromide.Spontaneous apoptosis and reactive oxygen species (ROS) generation were decreased in Rho cells. Following glycochenodeoxycholic acid (GCDCA) or paracetamol, Rho cells generated less ROS and were more resistant to cell death. Apoptosis induced by GCDCA and Fas was also reduced. The basal expression of Mdr1 was significantly enhanced, but this was not further stimulated by GCDCA or paracetamol, as observed in wild-type (WT) cells. Basal expression of Mrp1 and Mrp4 was similar in WT and Rho cells, whereas they were up-regulated only in WT cells after GCDCA or paracetamol, along with the transcription factors Shp and Nrf2, but not Fxr or Pxr. Increased expression of Nrf2 was accompanied by its enhanced nuclear translocation. Glycoursodeoxycholic acid failed to cause any of the effects observed for GCDCA or paracetamol.The Nrf2-mediated pathway is partly independent of ROS production. Nuclear translocation of Nrf2 is insufficient to up-regulate Mdr1, Mrp1 and Mrp4, which requires the participation of other regulatory element(s) whose activation in response to GCDCA and paracetamol is impaired in Rho cells and hence probably sensitive to ROS.

Carboxylesterases (CEs) metabolize a wide range of xenobiotic substrates including heroin, cocaine, meperidine and the anticancer agent CPT-11. In this study, we have purified to homogeneity human liver and intestinal CEs and compared their ability with hydrolyse heroin, cocaine and CPT-11.The hydrolysis of heroin and cocaine by recombinant human CEs was evaluated and the kinetic parameters determined. In addition, microsomal samples prepared from these tissues were subjected to chromatographic separation, and substrate hydrolysis and amounts of different CEs were determined.In contrast to previous reports, cocaine was not hydrolysed by the human liver CE, hCE1 (CES1), either as highly active recombinant protein or as CEs isolated from human liver or intestinal extracts. These results correlated well with computer-assisted molecular modelling studies that suggested that hydrolysis of cocaine by hCE1 (CES1), would be unlikely to occur. However, cocaine, heroin and CPT-11 were all substrates for the intestinal CE, hiCE (CES2), as determined using both the recombinant protein and the tissue fractions. Again, these data were in agreement with the modelling results.These results indicate that the human liver CE is unlikely to play a role in the metabolism of cocaine and that hydrolysis of this substrate by this class of enzymes is via the human intestinal protein hiCE (CES2). In addition, because no enzyme inhibition is observed at high cocaine concentrations, potentially this route of hydrolysis is important in individuals who overdose on this agent.

We aimed to demonstrate the involvement of 5-HT(1A) receptors in the therapeutic effect of cannabidiol, a non-psychoactive constituent of Cannabis sativa, in a model of hepatic encephalopathy induced by bile-duct ligation (BDL) in mice.Cannabidiol (5 mg x kg(-1); i.p.) was administered over 4 weeks to BDL mice. Cognition and locomotion were evaluated using the eight-arm maze and the open field tests respectively. Hippocampi were analysed by RT-PCR for expression of the genes for tumour necrosis factor-alpha receptor 1, brain-derived neurotrophic factor (BDNF) and 5-HT(1A) receptor. N-(2-(4-(2-methoxy-phenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide (WAY-100635), a 5-HT(1A) receptor antagonist (0.5 mg x kg(-1)), was co-administered with cannabidiol. Liver function was evaluated by measuring plasma liver enzymes and bilirubin.Cannabidiol improved cognition and locomotion, which were impaired by BDL, and restored hippocampal expression of the tumour necrosis factor-alpha receptor 1 and the BDNF genes, which increased and decreased, respectively, following BDL. It did not affect reduced 5-HT(1A) expression in BDL mice. All the effects of cannabidiol, except for that on BDNF expression, were blocked by WAY-100635, indicating 5-HT(1A) receptor involvement in cannabidiol's effects. Cannabidiol did not affect the impaired liver function in BDL.The behavioural outcomes of BDL result from both 5-HT(1A) receptor down-regulation and neuroinflammation. Cannabidiol reverses these effects through a combination of anti-inflammatory activity and activation of this receptor, leading to improvement of the neurological deficits without affecting 5-HT(1A) receptor expression or liver function. BDNF up-regulation by cannabidiol does not seem to account for the cognitive improvement.

1. OPC-31260, a benzazepine derivative, has been studied for its ability to antagonize the binding of arginine vasopressin (AVP) to receptors in rat liver (V1) and kidney (V2) plasma membranes, for antagonism of the antidiuretic action of AVP in alcohol-anaesthetized rats and for diuretic action in conscious normal rats. 2. OPC-31260 caused a competitive displacement of [3H]-AVP binding to both V1 and V2 receptors with IC50 values of 1.2 +/- 0.2 x 10(-6) M and 1.4 +/- 0.2 x 10(-8) M, respectively. 3. OPC-31260 at doses of 10 to 100 micrograms kg-1, i.v., inhibited the antidiuretic action of exogenously administered AVP in water-loaded, alcohol-anaesthetized rats in a dose-dependent manner. OPC-31260 did not exert an antidiuretic activity suggesting that it is not a partial V2 receptor agonist. 4. After oral administration at doses of 1 to 30 mg kg-1 in normal conscious rats, OPC-31260 dose-dependently increased urine flow and decreased urine osmolality. The diuretic action of OPC-31260 was characterized as aquaresis, the mode of diuretic action being different from previously known diuretic agents such as furosemide, hydrochlorothiazide and spironolactone. 5. The results indicate that OPC-31260 is a selective V2 receptor antagonist and behaves as an aquaretic agent. OPC-31260 will be a useful tool in studying the physiological role of AVP and in the treatment of various conditions characterized by water retention.

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide expressed in the lateral hypothalamus that is involved in feeding and body weight regulation. Intracerebroventricular infusion of a peptidic MCH1 receptor antagonist ameliorated obesity in murine models. Recently, small molecule MCH1 receptor antagonists have been developed and characterized for the treatment of obesity. However, little is known of the mechanism of the anti-obesity effects of MCH1 receptor antagonists.To examine the mechanisms of action of the anti-obesity effect of MCH1 receptor antagonists more precisely, we conducted a pair-feeding study in mice with diet-induced obesity (DIO), chronically treated with an orally active and highly selective MCH1 receptor antagonist and examined changes in mRNA expression levels in liver, brown and white adipose tissues. We also assessed the acute effects of the MCH1 receptor antagonist in energy expenditure under thermoneutral conditions.Treatment with the MCH1 receptor antagonist at 30 mg.kg(-1) for 1 month moderately suppressed feeding and significantly reduced body weight by 24%. In contrast, pair-feeding resulted in a smaller weight reduction of 10%. Treatment with the MCH1 receptor antagonist resulted in a higher body temperature compared with the pair-fed group. TaqMan and calorimetry data suggested that the MCH1 receptor antagonist also stimulated thermogenesis.Our results indicate that an MCH1 receptor antagonist caused anti-obesity effects im mice by acting on both energy intake and energy expenditure.

(-)-Stepholidine has high affinity for dopamine D(1) and D(2) receptors. The aims of the present study were to examine the oral bioavailability and brain penetration of (-)-stepholidine and to gain understanding of mechanisms governing its transport across the enterohepatic barrier and the blood-brain barrier.The pharmacokinetics of (-)-stepholidine was studied in rats and microdialysis was used to measure delivery to the brain. These studies were supported by biological measurement of unbound (-)-stepholidine. Membrane permeability was assessed using Caco-2 cell monolayers. Metabolite profiling of (-)-stepholidine in rat bile and plasma was performed. Finally, in vitro metabolic stability and metabolite profile of (-)-stepholidine were examined to compare species similarities and differences between rats and humans.Orally administered (-)-stepholidine was rapidly absorbed from the gastrointestinal tract; two plasma concentration peaks were seen, and the second peak might result from enterohepatic circulation. Due to extensive pre-systemic metabolism, the oral bioavailability of (-)-stepholidine was poor (2%). However, the compound was extensively transported across the blood-brain barrier, demonstrating an AUC (area under concentration-time curve) ratio of brain : plasma of approximately 0.7. (-)-Stepholidine showed good membrane permeability that was unaffected by P-glycoprotein and multidrug resistance-associated protein 2. In vitro (-)-stepholidine was metabolized predominantly by glucuronidation and sulphation in rats and humans, but oxidation of this substrate was very low.Although (-)-stepholidine exhibits good brain penetration, future development efforts should aim at improving its oral bioavailability by protecting against pre-systemic glucuronidation or sulphation. In this regard, prodrug approaches may be useful.

Hepatic encephalopathy is a neuropsychiatric syndrome caused by liver failure. In view of the effects of cannabinoids in a thioacetamide-induced model of hepatic encephalopathy and liver disease and the beneficial effect of capsaicin (a TRPV1 agonist) in liver disease, we assumed that capsaicin may also affect hepatic encephalopathy.Fulminant hepatic failure was induced in mice by thioacetamide and 24 h later, the animals were injected with one of the following compound(s): 2-arachidonoylglycerol (CB(1), CB(2) and TRPV1 receptor agonist); HU308 (CB(2) receptor agonist), SR141716A (CB(1) receptor antagonist); SR141716A+2-arachidonoylglycerol; SR144528 (CB(2) receptor antagonist); capsaicin; and capsazepine (TRPV1 receptor agonist and antagonist respectively). Their neurological effects were evaluated on the basis of activity in the open field, cognitive function in an eight-arm maze and a neurological severity score. The mice were killed 3 or 14 days after thioacetamide administration. 2-arachidonoylglycerol and 5-hydroxytryptamine (5-HT) levels were determined by gas chromatography-mass spectrometry and high-performance liquid chromatography with electrochemical detection, respectively.Capsaicin had a neuroprotective effect in this animal model as shown by the neurological score, activity and cognitive function. The effect of capsaicin was blocked by capsazepine. Thioacetamide induced astrogliosis in the hippocampus and the cerebellum and raised brain 5-hydroxytryptamine levels, which were decreased by capsaicin, SR141716A and HU-308. Thioacetamide lowered brain 2-arachidonoylglycerol levels, an effect reversed by capsaicin.Capsaicin improved both liver and brain dysfunction caused by thioacetamide, suggesting that both the endocannabinoid and the vanilloid systems play important roles in hepatic encephalopathy. Modulation of these systems may have therapeutic value.

The lipid phosphatase known as SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) plays an important role in the regulation of the intracellular insulin signalling pathway. Recent studies have suggested that inhibition of SHIP2 could produce significant benefits in treatment of type 2 diabetes. However, there were no small molecule SHIP2 inhibitors and we, therefore, aimed to identify this type of compound.The phosphatase assay with malachite green was used for high-throughput screening. The pharmacological profiles of suitable compounds were further characterized in phosphatase assays, cellular assays and oral administration in normal and diabetic (db/db) mice.During high-throughput screening, AS1949490 was identified as a potent SHIP2 inhibitor (IC(50)= 0.62 microM for SHIP2). This compound was also selective for SHIP2 relative to other intracellular phosphatases. In L6 myotubes, AS1949490 increased the phosphorylation of Akt, glucose consumption and glucose uptake. In FAO hepatocytes, AS1949490 suppressed gluconeogenesis. Acute administration of AS1949490 inhibited the expression of gluconeogenic genes in the livers of normal mice. Chronic treatment of diabetic db/db mice with AS1949490 significantly lowered the plasma glucose level and improved glucose intolerance. These in vivo effects were based in part on the activation of intracellular insulin signalling pathways in the liver.This is the first report of a small molecule inhibitor of SHIP2. This compound will help to elucidate the physiological functions of SHIP2 and its involvement in various diseases, such as type 2 diabetes.