The Impact of the Drying Process on the Antioxidant and Anti-Inflammatory Potential of Dried Ripe Coffee Cherry Pulp Soluble Powder.

Coffee fruit cascara, which is the skin and pulp of the coffee cherry, has been authorized as a novel food for commercialization in the European Union. The present research assessed the feasibility of using spray drying to produce a soluble powder called instant cascara (IC), employing sun-dried ripe coffee cherry pulp as a raw material. Although there were no significant differences (p > 0.05) in the overall antioxidant capacity between the freeze-dried and spray-dried samples, after an in vitro simulation of the digestion process, the spray-dried sample was significantly (p < 0.05) more antioxidant. Both samples reduced physiological intracellular ROS and significantly decreased (p < 0.05) the secretion of the pro-inflammatory factor NO. Alkaloids and phenolic compounds were detected in intestinal digests. In conclusion, spray drying is a good technique for producing IC as its use does not affect its properties and causes less environmental impact than freeze drying, as calculated by life cycle assessment. Sensory analysis did not show significant differences between the commercial beverage and the IC beverage in the adult population. IC at 10 mg/mL was significantly less accepted in adolescents than the commercial beverage. Future work will include the reformulation of the IC beverage at 10 mg/mL, which has antioxidant and anti-inflammatory potential, to increase its hedonic acceptance in all consumer segments.

Role for PPARγ in obesity-induced hepatic steatosis as determined by hepatocyte- and macrophage-specific conditional knockouts.

Peroxisome proliferator-activated receptor (PPAR) γ is a nuclear receptor central to glucose and lipid homeostasis. PPARγ role in nonalcoholic fatty liver disease is controversial because PPARγ overexpression is a general property of steatotic livers, but its activation by thiazolidinediones reduces hepatic steatosis. Here, we investigated hepatic PPARγ function by using Cre-loxP technology to generate hepatocyte (PPARγ(Δhep))- and macrophage (PPARγ(Δmac))-specific PPARγ-knockout mice. Targeted deletion of PPARγ in hepatocytes, and to a lesser extent in macrophages, protected mice against high-fat diet-induced hepatic steatosis. Down-regulated expression of genes involved in lipogenesis (SCD1, SREBP-1c, and ACC), lipid transport (CD36/FAT, L-FABP, and MTP), and ß-oxidation (PPARα and ACO) was observed in PPARγ(Δhep) mice. Moreover, PPARγ(Δhep) mice showed improved glucose tolerance and reduced PEPCK expression without changes in Pcx, Fbp1, and G6Pc expression and CREB and JNK phosphorylation. In precision-cut liver slices (PCLSs) and hepatocytes, rosiglitazone either alone or in combination with oleic acid increased triglyceride accumulation, an effect that was blocked by the PPARγ antagonist biphenol A diglycidyl ether (BADGE). PCLSs and hepatocytes from PPARγ(Δhep) mice showed blunted responses to rosiglitazone and oleic acid, whereas the response to these compounds remained intact in PCLSs from PPARγ(Δmac) mice. Collectively, these findings establish PPARγ expression in hepatocytes as a prosteatotic factor in fatty liver disease.

5-lipoxygenase activating protein signals adipose tissue inflammation and lipid dysfunction in experimental obesity.

The presence of the so-called low-grade inflammatory state is recognized as a critical event in adipose tissue dysfunction, leading to altered secretion of adipokines and free fatty acids (FFAs), insulin resistance, and development of hepatic complications associated with obesity. This study was designed to investigate the potential contribution of the proinflammatory 5-lipoxygenase (5-LO) pathway to adipose tissue inflammation and lipid dysfunction in experimental obesity. Constitutive expression of key components of the 5-LO pathway, as well as leukotriene (LT) receptors, was detected in adipose tissue as well as in adipocyte and stromal vascular fractions. Adipose tissue from obese mice, compared with that from lean mice, exhibited increased 5-LO activating protein (FLAP) expression and LTB(4) levels. Incubation of adipose tissue with 5-LO products resulted in NF-kappaB activation and augmented secretion of proinflammatory adipokines such as MCP-1, IL-6, and TNF-alpha. In addition, LTB(4), but not LTD(4), reduced FFA uptake in primary adipocytes, whereas 5-LO inhibition suppressed isoproterenol-induced adipose tissue lipolysis. In mice with dietary obesity, elevated FLAP expression in adipose tissue was paralleled with macrophage infiltration, increased circulating FFA levels, and hepatic steatosis, phenomena that were reversed by FLAP inhibition with Bay-X-1005. Interestingly, FLAP inhibition induced AMP-activated protein kinase phosphorylation in parallel with decreases in hormone-sensitive lipase activity and the expression and secretion of TNF-alpha and IL-6. Similar effects were observed in differentiated 3T3-L1 adipocytes incubated with either Bay-X-1005 or the selective LTB(4) receptor antagonist U-75302. Taken together, these findings indicate that the 5-LO pathway signals the adipose tissue low-grade inflammatory state and steatogenic potential in experimental obesity.

Enhancement of the Antioxidant Capacity of Thyme and Chestnut Honey by Addition of Bee Products.

Honey consumption and imports have increased in recent years, and it is considered by consumers to be a healthy alternative to more commonly used sweeteners. Honey contains a mixture of polyphenols and antioxidant compounds, and the botanical origin and geographical area of collection play an important role on its chemical composition. The present study investigated the physicochemical properties, total phenolic content and antioxidant capacity of Spanish thyme honey and chestnut honey, and their mixtures with royal jelly (2% and 10%) and propolis (2% and 10%). The analysis of the physicochemical parameters of both honey samples showed values within the established limits. Propolis showed the highest value of total phenolic content (17.21-266.83 mg GAE/100 g) and antioxidant capacity (DPPH, ORAC and ABTS assays; 0.63-24.10 µg eq. Tx/g, 1.61-40.82 µg eq. Tx/g and 1.89-68.54 µg eq. Tx/g, respectively), and significantly reduced ROS production in human hepatoma cells. In addition, mixtures of honey with 10% of propolis improved the results obtained with natural honey, increasing the value of total phenolic content and antioxidant capacity. A significant positive correlation was observed between total phenolic compounds and antioxidant capacity. Therefore, the antioxidant capacity could be attributed to the phenolic compounds present in the samples, at least partially. In conclusion, our results indicated that thyme and chestnut honey supplemented with propolis can be an excellent natural source of antioxidants and could be incorporated as a potential food ingredient with biological properties of technological interest, added as a preservative. Moreover, these mixtures could be used as natural sweeteners enriched in antioxidants and other bioactive compounds.

5-lipoxygenase deficiency reduces hepatic inflammation and tumor necrosis factor alpha-induced hepatocyte damage in hyperlipidemia-prone ApoE-null mice.

UNLABELLED: The actual risk factors that drive hepatic inflammation during the transition from steatosis to steatohepatitis are unknown. We recently demonstrated that hyperlipidemia-prone apolipoprotein E-deficient (ApoE(-/-)) mice exhibit hepatic steatosis and increased susceptibility to hepatic inflammation and advanced fibrosis. Because the proinflammatory 5-lipoxygenase (5-LO) pathway was found to be up-regulated in these mice and given that 5-LO deficiency confers cardiovascular protection to ApoE(-/-) mice, we determined the extent to which the absence of 5-LO would alter liver injury in these mice. Compared with ApoE(-/-) mice, which showed expected hepatic steatosis and inflammation, ApoE/5-LO double-deficient (ApoE(-/-)/5-LO(-/-)) mice exhibited reduced hepatic inflammation, macrophage infiltration, tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1) and interleukin (IL)-18 expression, caspase-3 and nuclear factor-kappaB (NF-kappaB) activities, and serum alanine aminotransferase levels in the absence of changes in hepatic steatosis. The lack of 5-LO produced a remarkable insulin-sensitizing effect in the adipose tissue because peroxisome proliferator-activated receptor gamma, insulin receptor substrate-1, and adiponectin were up-regulated, whereas c-Jun amino-terminal kinase phosphorylation and MCP-1 and IL-6 expression were down-regulated. On the other hand, hepatocytes isolated from ApoE(-/-)/5-LO(-/-) mice were more resistant to TNF-alpha-induced apoptosis. The 5-LO products leukotriene (LT) B(4), LTD(4), and 5-HETE consistently triggered TNF-alpha-induced apoptosis and compromised hepatocyte survival by suppressing NF-kappaB activity in the presence of actinomycin D. Moreover, ApoE(-/-)/5-LO(-/-) mice were protected against sustained high-fat diet (HFD)-induced liver injury and hepatic inflammation, macrophage infiltration and insulin resistance were significantly milder than those of ApoE(-/-) mice. Finally, pharmacological inhibition of 5-LO significantly reduced hepatic inflammatory infiltrate in the HFD and ob/ob models of fatty liver disease. CONCLUSION: These combined data indicate that hyperlipidemic mice lacking 5-LO are protected against hepatic inflammatory injury, suggesting that 5-LO is involved in mounting hepatic inflammation in metabolic disease.

Obesity-induced insulin resistance and hepatic steatosis are alleviated by omega-3 fatty acids: a role for resolvins and protectins.

Omega-3-polyunsaturated fatty acids (omega-3-PUFAs) have well-documented protective effects that are attributed not only to eicosanoid inhibition but also to the formation of novel biologically active lipid mediators (i.e., resolvins and protectins). In this study, we examined their effects on ob/ob mice, an obesity model of insulin resistance and fatty liver disease. Dietary intake of omega-3-PUFAs had insulin-sensitizing actions in adipose tissue and liver and improved insulin tolerance in obese mice. Genes involved in insulin sensitivity (PPARgamma), glucose transport (GLUT-2/GLUT-4), and insulin receptor signaling (IRS-1/IRS-2) were up-regulated by omega-3-PUFAs. Moreover, omega-3-PUFAs increased adiponectin, an anti-inflammatory and insulin-sensitizing adipokine, and induced AMPK phosphorylation, a fuel-sensing enzyme and a gatekeeper of the energy balance. Concomitantly, hepatic steatosis was alleviated by omega-3-PUFAs. A lipidomic analysis with liquid chromatography/mass spectrometry/mass spectrometry revealed that omega-3-PUFAs inhibited the formation of omega-6-PUFA-derived eicosanoids, while triggering the formation of omega-3-PUFA-derived resolvins and protectins. Moreover, representative members of these lipid mediators, namely resolvin E1 and protectin D1, mimicked the insulin-sensitizing and antisteatotic effects of omega-3-PUFAs and induced adiponectin expression to a similar extent that of rosiglitazone, a member of the thiazolidinedione family of antidiabetic drugs. Taken together, these findings uncover beneficial actions of omega-3-PUFAs and their bioactive lipid autacoids in preventing obesity-induced insulin resistance and hepatic steatosis.

Protection from hepatic lipid accumulation and inflammation by genetic ablation of 5-lipoxygenase.

Five-lipoxygenase (5-LO) has been postulated as a pathogenic factor in liver injury. Indeed, Alox5, the gene coding for 5-LO, is heavily over-expressed in experimental liver disease, in which 5-LO inhibition consistently ameliorates hepatic steatosis, inflammation and fibrosis. Herein, we report the findings in mice with targeted deletion of Alox5 as a proof of concept of the role of 5-LO in liver injury. Our findings demonstrate that ablation of Alox5 in mice confers protection against carbon tetrachloride-induced liver injury since hepatic necroinflammation, inflammatory infiltrate, hepatocyte ballooning and serum ALT levels were significantly reduced in Alox5-deficient mice. These mice also showed a lower degree of hepatic steatosis, which affected micro- and macrosteatosis to a similar extent. Moreover, microarray analysis revealed a differential profile of hepatic gene expression in Alox5-deficient mice, with a total of 117 genes differentially expressed in these animals. Functional grouping of these genes revealed that 28 (approximately 24% of total changes) were related to the category of lipid metabolism, including the lipogenic factors Lpin1, C/EBP, Fasn, Acly and Elovl6. Moreover, Ingenuity Pathway Analysis revealed lipid metabolism as the molecular/cellular function most affected by the loss of Alox5. These findings confirm at a genetic level that Alox5 plays a pathogenic role in the response of the liver to injury.

Comparative protection against liver inflammation and fibrosis by a selective cyclooxygenase-2 inhibitor and a nonredox-type 5-lipoxygenase inhibitor.

In this study, we examined the relative contribution of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO), two major proinflammatory pathways up-regulated in liver disease, to the progression of hepatic inflammation and fibrosis. Separate administration of 4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-236), a selective COX-2 inhibitor, and CJ-13,610, a 5-LO inhibitor, to carbon tetrachloride-treated mice significantly reduced fibrosis as revealed by the analysis of Sirius Red-stained liver sections without affecting necroinflammation. Conversely, combined administration of SC-236 and 4-[3-[4-(2-methylimidazol-1-yl)-phenylthio]]phenyl-3,4,5,6-tetrahydro-2H-pyran-4-carboxamide (CJ-13,610) reduced both necroinflammation and fibrosis. These findings were confirmed in 5-LO-deficient mice receiving SC-236, which also showed reduced hepatic monocyte chemoattractant protein 1 expression. Interestingly, SC-236 and CJ-13,610 significantly increased the number of nonparenchymal liver cells with apoptotic nuclei (terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive). Additional pharmacological profiling of SC-236 and CJ-13,610 was performed in macrophages, the primary hepatic inflammatory cell type. In these cells, SC-236 inhibited prostaglandin (PG) E2 formation in a concentration-dependent manner, whereas CJ-13,610 blocked leukotriene B4 biosynthesis. Of note, the simultaneous addition of SC-236 and CJ-13,610 resulted in a higher inhibitory profile on PGE2 biosynthesis than the dual COX/5-LO inhibitor licofelone. These drugs differentially regulated interleukin-6 mRNA expression in macrophages. Taken together, these findings indicate that both COX-2 and 5-LO pathways are contributing factors to hepatic inflammation and fibrosis and that these two pathways of the arachidonic acid cascade represent potential targets for therapy.

Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA.

Docosahexaenoic acid (DHA) is a omega-3 essential fatty acid that reduces the incidence and severity of a number of diseases. Recently, a novel series of DHA-derived lipid mediators with potent protective actions has been identified. In this study we demonstrate that dietary amplification of these DHA-derived products protects the liver from necroinflammatory injury. In vitro, supplementation of hepatocytes with DHA significantly reduced hydrogen peroxide-induced DNA damage, evaluated by the "comet assay," and oxidative stress, determined by measurement of malondialdehyde levels. In vivo, dietary supplementation of mice with DHA ameliorated carbon tetrachloride-induced necroinflammatory damage. In addition, hepatic cyclooxygenase-2 expression and PGE2 levels were significantly reduced in mice fed DHA-enriched diets. In these animals, increased hepatic formation of DHA-derived lipid mediators (i.e., 17S-hydroxy-DHA (17S-HDHA) and protectin D1) was detected by HPLC-gas chromatography/mass spectrometry analysis. Consistent with these findings, synthetic 17-HDHA abrogated genotoxic and oxidative damage in hepatocytes and decreased TNF-alpha release and 5-lipoxygenase expression in macrophages. In a transactivation assay, 17-HDHA acted in a concentration-dependent manner as a PPARgamma agonist. Taken together, these findings identify a potential role for DHA-derived products, specifically 17S-HDHA and protectin D1, in mediating the protective effects of dietary DHA in necroinflammatory liver injury.

The selective cyclooxygenase-2 inhibitor SC-236 reduces liver fibrosis by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation.

The importance of inflammation in initiating the sequence of events that lead to liver fibrosis is increasingly recognized. In this study, we tested the effects of SC-236, a selective cyclooxygenase (COX)-2 inhibitor, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Livers from CCl4-treated rats showed increased COX-2 expression and 15-deoxy-prostaglandin (PG)J2 (15d-PGJ2) formation, as well as decreased peroxisome proliferator-activated receptor (PPAR)gamma expression. In these animals, SC-236 reduced liver fibrosis as revealed by histological analysis and by a reduction in hepatic hydroxyproline levels, metalloproteinase-2 activity, and alpha-smooth muscle actin expression. Interestingly, SC-236 normalized 15d-PGJ2 levels and restored PPARgamma expression in the liver of CCl4-treated rats. In isolated hepatic stellate cells (HSCs)--the major player in liver fibrogenesis--and Kupffer cells--the cell type primarily responsible for increased hepatic COX-2-SC-236 exhibited remarkable pro-apoptotic and growth inhibitory properties. Of interest, SC-236 decreased HSC viability to a similar extent than the PPARgamma ligand rosiglitazone. Moreover, SC-236 significantly induced PPARgamma expression in HSCs and acted as a potent PPARgamma agonist in a luciferase-reporter trans-activation assay. These data indicate that, by mechanisms involving non-parenchymal cell apoptosis and PPARgamma activation, the selective COX-2 inhibitor SC-236 might have therapeutic potential for prevention of liver fibrosis.

Inhibition of 5-lipoxygenase-activating protein abrogates experimental liver injury: role of Kupffer cells.

Activation of Kupffer cells is a prominent feature of necro-inflammatory liver injury. We have recently demonstrated that 5-lipoxygenase (5-LO) and its accessory protein, 5-LO-activating protein (FLAP), are essential for the survival of Kupffer cells in culture, as their inhibition drives these liver resident macrophages to programmed cell death. In the current study, we explored whether the potent FLAP inhibitor, Bay-X-1005, reduces the number of Kupffer cells in vivo and whether this pharmacological intervention protects the liver from carbon tetrachloride (CCl(4))-induced damage. Rats treated with CCl(4) showed an increased number of Kupffer cells, an effect that was abrogated by the administration of Bay-X-1005 (100 mg/Kg body weight, per oral, daily). Consistent with a role for Kupffer cells in necro-inflammatory liver injury, partial depletion of Kupffer cells following FLAP inhibition was associated with a remarkable hepatoprotective action. Indeed, Bay-X-1005 significantly reduced the intense hepatocyte degeneration and large bridging necrosis induced by CCl(4) treatment. Moreover, Bay-X-1005 induced a reduction in the gelatinolytic activity of matrix metalloproteinase-2 (MMP-2) and a decrease in mRNA expression of tissue inhibitor of MMP-2. The FLAP inhibitor reduced leukotriene (LT)B(4) and cysteinyl LT levels and down-regulated 5-LO and FLAP protein expression in the liver. It is interesting that a significant increase in the hepatic formation of lipoxin A(4), an endogenous, anti-inflammatory lipid mediator involved in the resolution of inflammation, was observed after the administration of Bay-X-1005. These findings support the concept that modulation of the 5-LO pathway by FLAP inhibition may be useful in the prevention of hepatotoxin-induced necro-inflammatory injury.

Aspirin (ASA) regulates 5-lipoxygenase activity and peroxisome proliferator-activated receptor alpha-mediated CINC-1 release in rat liver cells: novel actions of lipoxin A4 (LXA4) and ASA-triggered 15-epi-LXA4.

The mechanism of action of aspirin (ASA) is related to cyclooxygenase (COX) inhibition, but additional actions cannot be excluded for their antiinflammatory properties and antithrombotic activity. In the current investigation, we examined the effects of ASA on COX and 5-lipoxygenase (5-LO) pathways and its impact on peroxisome proliferator-activated receptor alpha (PPARalpha) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels in rat liver cells. In Kupffer cells, the liver resident macrophages, ASA switched eicosanoid biosynthesis from prostaglandin E2 (PGE2) to leukotriene B4 (LTB4) and 15-epi-lipoxin A4 (15-epi-LXA4) formation. In hepatocytes, ASA significantly inhibited PPARalpha protein expression and CINC-1 secretion, effects that were also observed in hepatocytes exposed to the selective PPARalpha agonist Wy-14643. In contrast, treatment of hepatocytes with PGE2 in association with LTB4 had no significant effect on PPARalpha but stimulated CINC-1 release. Interestingly, the endogenous antiinflammatory eicosanoids LXA4 and ASA-triggered 15-epi-LXA4, in addition to inhibiting macrophage 5-LO activity to a similar extent as PGE2, significantly reduced PPARalpha and CINC-1 levels in hepatocytes. Taken together and because arachidonic acid-derived products, PPARalpha levels, and CINC-1 secretion are involved in the extent and duration of an inflammatory response, these findings provide additional molecular mechanisms for the pharmacological properties of ASA.

Inhibition of 5-lipoxygenase induces cell growth arrest and apoptosis in rat Kupffer cells: implications for liver fibrosis.

The existence of an increased number of Kupffer cells is recognized as critical in the initiation of the inflammatory cascade leading to liver fibrosis. Because 5-lipoxygenase (5-LO) is a key regulator of cell growth and survival, in the current investigation we assessed whether inhibition of the 5-LO pathway would reduce the excessive number of Kupffer cells and attenuate inflammation and fibrosis in experimental liver disease. Kupffer cells were the only liver cell type endowed with a metabolically active 5-LO pathway (i.e., expressed mRNAs for 5-LO, 5-LO-activating protein [FLAP], and leukotriene [LT] C4 synthase and generated LTB4 and cysteinyl-LTs). Both the selective 5-LO inhibitor AA861 and the FLAP inhibitor BAY-X-1005 markedly reduced the number of Kupffer cells in culture. The antiproliferative properties of AA861 and BAY-X-1005 were associated with the occurrence of condensed nuclei, fragmented DNA, and changes in DNA content and cell cycle frequency distribution consistent with an apoptotic process. In vivo, in carbon tetrachloride-treated rats, BAY-X-1005 had a significant antifibrotic effect and reduced liver damage and the hepatic content of hydroxyproline. Together, these findings indicate a novel mechanism by which inactivation of the 5-LO pathway could disrupt the sequence of events leading to liver inflammation and fibrosis.

Cyclooxygenase-1 derived prostaglandins are involved in the maintenance of renal function in rats with cirrhosis and ascites.

1. The maintenance of renal function in decompensated cirrhosis is highly dependent on prostaglandins (PGs). Since PG synthesis is mediated by cyclooxygenase-1 and -2 (COX-1 and COX-2), the present study was designed to examine which COX isoform is involved in this phenomenon. 2. Renal COX-1 and COX-2 protein expression and distribution were analysed by Western blot and immunohistochemistry in nine rats with carbon tetrachloride-induced cirrhosis and ascites and 10 control animals. The effects of placebo and selective COX-1 (SC-560) and COX-2 (celecoxib) inhibitors on urine flow (V), urinary excretion of sodium (U(Na)V) and PGE(2) (U(PGE2)V), glomerular filtration rate (GFR), renal plasma flow (RPF), the diuretic and natriuretic responses to furosemide and renal water metabolism were assessed in 88 rats with cirrhosis and ascites. 3. COX-1 protein levels were found to be unchanged in kidneys from cirrhotic rats. In contrast, these animals showed enhanced renal COX-2 protein expression which was focally increased in the corticomedullary region. Although U(PGE2)V was equally reduced by SC-560 and celecoxib, only SC-560 produced a significant decrease in U(Na)V, GFR and RPF and a pronounced impairment in the diuretic and natriuretic responses to furosemide in rats with cirrhosis and ascites. Neither SC-560 nor celecoxib affected renal water metabolism in cirrhotic rats. 4. These results indicate that despite abundant renal COX-2 protein expression, the maintenance of renal function in cirrhotic rats is mainly dependent on COX-1-derived prostaglandins.

Increased susceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential involvement of oxysterols.

The contribution of metabolic factors to the severity of liver disease is not completely understood. In this study, apolipoprotein E-deficient (ApoE-/-) mice were evaluated to define potential effects of hypercholesterolemia on the severity of carbon tetrachloride (CCl4)-induced liver injury. Under baseline conditions, hypercholesterolemic ApoE-/- mice showed increased hepatic oxidative stress (SOD activity/4-hydroxy-2-nonenal immunostaining) and higher hepatic TGF-beta1, MCP-1, and TIMP-1 expression than wild-type control mice. After CCl4 challenge, ApoE-/- mice exhibited exacerbated steatosis (Oil Red O staining), necroinflammation (hematoxylin-eosin staining), macrophage infiltration (F4/80 immunohistochemistry), and fibrosis (Sirius red staining and alpha-smooth muscle actin immunohistochemistry) and more severe liver injury [alanine aminotransferase (ALT) and aspartate aminotransferase] than wild-type controls. Direct correlations were identified between serum cholesterol and hepatic steatosis, fibrosis, and ALT levels. These changes did not reflect the usual progression of the disease in ApoE-/- mice, since exacerbated liver injury was not present in untreated age-paired ApoE-/- mice. Moreover, hepatic cytochrome P-450 expression was unchanged in ApoE-/- mice. To explore potential mechanisms, cell types relevant to liver pathophysiology were exposed to selected cholesterol-oxidized products. Incubation of hepatocytes with a mixture of oxysterols representative of those detected by GC-MS in livers from ApoE-/- mice resulted in a concentration-dependent increase in total lipoperoxides and SOD activity. In hepatic stellate cells, oxysterols increased IL-8 secretion through a NF-kappaB-independent mechanism and upregulated TIMP-1 expression. In macrophages, oxysterols increased TGF-beta1 secretion and MCP-1 expression in a concentration-dependent manner. Oxysterols did not compromise cell viability. Taken together, these findings demonstrate that hypercholesterolemic mice are sensitized to liver injury and that cholesterol-derived products (i.e., oxysterols) are able to induce proinflammatory and profibrogenic mechanisms in liver cells.

Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice.

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.

Gene expression profiling of renal dysfunction in rats with experimental cirrhosis.

BACKGROUND/AIMS: Renal dysfunction is a frequent complication in advanced cirrhosis. The mechanisms underlying this complication have classically been addressed through conventional methods of study of candidate genes, but never on a genome-wide scale. In this investigation, we used microarrays to monitor global gene expression changes in the kidney of cirrhotic rats. METHODS: Renal samples were obtained from control and carbon tetrachloride-induced cirrhotic rats. RNA samples were reverse-transcribed into Cy5-labeled cDNA, combined with a Cy3-labeled reference and hybridized to oligonucleotide microarrays. Microarrays were scanned in a Genepix 4000B and data analyzed by Luminator v2.0 software. RESULTS: A total of 620 genes were differentially regulated (354 up and 266 down) in the cirrhotic kidney, accounting for approximately 11% of all analyzed transcripts. Functional grouping of these genes revealed that 47 were related to the category of vascular tone and 85 to transporters/channels. Among these, we identified genes and pathways already associated with renal dysfunction as well as a new subset of genes previously unknown to participate in this complication, including a G protein-coupled receptor that binds apelin, a protein phosphatase (calcineurin B) and a number of neuropeptide receptors and growth factors. CONCLUSIONS: These findings furnish new data for mechanistic investigation into renal dysfunction in cirrhosis.

The selective cyclooxygenase-2 inhibitor celecoxib modulates the formation of vasoconstrictor eicosanoids and activates PPARgamma. Influence of albumin.

BACKGROUND/AIMS: Selective cyclooxygenase (COX)-2 inhibitors do not adversely affect renal function in experimental cirrhosis. In the current study, we investigated the molecular mechanisms underlying the effects of the selective COX-2 inhibitor, celecoxib, and assessed the influence of albumin on its actions. METHODS: Rat mesangial cells (RMC) were incubated with celecoxib in the absence or presence of albumin, and levels of selected vasoconstrictor eicosanoids, renin release and alpha-smooth muscle actin (alpha-SMA) expression were determined. The effects of celecoxib on PPARgamma were assessed in RMC co-transfected with PPARgamma and luciferase reporter constructs. RESULTS: Under resting conditions, RMC expressed COX-1, COX-2 and 12/15-lipoxygenase and mainly generated prostaglandin (PG)E2, thromboxane (TX)B2, 12-hydroxyeicosatetraenoic acid (12-HETE) and 8-epi-PGF2alpha. Celecoxib, in addition to reducing PGE2, significantly decreased 8-epi-PGF2alpha formation. In the presence of albumin, celecoxib also reduced TXB2 and 12-HETE. Albumin per se inhibited PGE2 as well as renin release. In trans-activation assays, celecoxib acted as a PPARgamma agonist whereas albumin inhibited PPARgamma as well as 15d-PGJ2-induced PPARgamma activation. Finally, celecoxib and albumin potentiated the inhibitory effect of 15d-PGJ2 on alpha-SMA expression. CONCLUSIONS: These data provide novel molecular mechanisms of celecoxib and their modulation by albumin, that may be relevant to prevent renal dysfunction in conditions of unbalanced effective blood volume.