Knock-in mice expressing a humanized arachidonic acid 15-lipoxygenase (Alox15) carry a partly dysfunctional erythropoietic system.

Arachidonic acid 15-lipoxygenases (ALOX15) play a role in mammalian erythropoiesis but they have also been implicated in inflammatory processes. Seven intact Alox genes have been detected in the mouse reference genome and the mouse Alox15 gene is structurally similar to the orthologous genes of other mammals. However, mouse and human ALOX15 orthologs have different functional characteristics. Human ALOX15 converts C20 polyenoic fatty acids like arachidonic acid mainly to the n-6 hydroperoxide. In contrast, the n-9 hydroperoxide is the major oxygenation product formed by mouse Alox15. Previous experiments indicated that Leu353Phe exchange in recombinant mouse Alox15 humanized the catalytic properties of the enzyme. To investigate whether this functional humanization might also work in vivo and to characterize the functional consequences of mouse Alox15 humanization we generated Alox15 knock-in mice (Alox15-KI), in which the Alox15 gene was modified in such a way that the animals express the arachidonic acid 15-lipoxygenating Leu353Phe mutant instead of the arachidonic acid 12-lipoxygenating wildtype enzyme. These mice develop normally, they are fully fertile but display modified plasma oxylipidomes. In young individuals, the basic hematological parameters were not different when Alox15-KI mice and outbred wildtype controls were compared. However, when growing older male Alox15-KI mice develop signs of dysfunctional erythropoiesis such as reduced hematocrit, lower erythrocyte counts and attenuated hemoglobin concentration. These differences were paralleled by an improved ex vivo osmotic resistance of the peripheral red blood cells. Interestingly, such differences were not observed in female individuals suggesting gender specific effects. In summary, these data indicated that functional humanization of mouse Alox15 induces defective erythropoiesis in aged male individuals.

Humanization of the Reaction Specificity of Mouse Alox15b Inversely Modified the Susceptibility of Corresponding Knock-In Mice in Two Different Animal Inflammation Models.

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in the pathogenesis of inflammatory diseases, and its pro- and anti-inflammatory effects have been reported for different ALOX-isoforms. Human ALOX15B oxygenates arachidonic acid to its 15-hydroperoxy derivative, whereas the corresponding 8-hydroperoxide is formed by mouse Alox15b (Alox8). This functional difference impacts the biosynthetic capacity of the two enzymes for creating pro- and anti-inflammatory eicosanoids. To explore the functional consequences of the humanization of the reaction specificity of mouse Alox15b in vivo, we tested Alox15b knock-in mice that express the arachidonic acid 15-lipoxygenating Tyr603Asp and His604Val double mutant of Alox15b, instead of the arachidonic acid 8-lipoxygenating wildtype enzyme, in two different animal inflammation models. In the dextran sodium sulfate-induced colitis model, female Alox15b-KI mice lost significantly more bodyweight during the acute phase of inflammation and recovered less rapidly during the resolution phase. Although we observed significant differences in the colonic levels of selected pro- and anti-inflammatory eicosanoids during the time-course of inflammation, there were no differences between the two genotypes at any time-point of the disease. In Freund's complete adjuvant-induced paw edema model, Alox15b-KI mice were less susceptible than outbred wildtype controls, though we did not observe significant differences in pain perception (Hargreaves-test, von Frey-test) when the two genotypes were compared. our data indicate that humanization of the reaction specificity of mouse Alox15b (Alox8) sensitizes mice for dextran sodium sulfate-induced experimental colitis, but partly protects the animals in the complete Freund's adjuvant-induced paw edema model.

The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism.

Reduced expression of the Indy ("I am Not Dead, Yet") gene in lower organisms promotes longevity in a manner akin to caloric restriction. Deletion of the mammalian homolog of Indy (mIndy, Slc13a5) encoding for a plasma membrane-associated citrate transporter expressed highly in the liver, protects mice from high-fat diet-induced and aging-induced obesity and hepatic fat accumulation through a mechanism resembling caloric restriction. We studied a possible role of mIndy in human hepatic fat metabolism. In obese, insulin-resistant patients with nonalcoholic fatty liver disease, hepatic mIndy expression was increased and mIndy expression was also independently associated with hepatic steatosis. In nonhuman primates, a 2-year high-fat, high-sucrose diet increased hepatic mIndy expression. Liver microarray analysis showed that high mIndy expression was associated with pathways involved in hepatic lipid metabolism and immunological processes. Interleukin-6 (IL-6) was identified as a regulator of mIndy by binding to its cognate receptor. Studies in human primary hepatocytes confirmed that IL-6 markedly induced mIndy transcription through the IL-6 receptor and activation of the transcription factor signal transducer and activator of transcription 3, and a putative start site of the human mIndy promoter was determined. Activation of the IL-6-signal transducer and activator of transcription 3 pathway stimulated mIndy expression, enhanced cytoplasmic citrate influx, and augmented hepatic lipogenesis in vivo. In contrast, deletion of mIndy completely prevented the stimulating effect of IL-6 on citrate uptake and reduced hepatic lipogenesis. These data show that mIndy is increased in liver of obese humans and nonhuman primates with NALFD. Moreover, our data identify mIndy as a target gene of IL-6 and determine novel functions of IL-6 through mINDY. CONCLUSION: Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450. (Hepatology 2017;66:616-630).

Insulin-induced cytokine production in macrophages causes insulin resistance in hepatocytes.

Overweight and obesity are associated with hyperinsulinemia, insulin resistance, and a low-grade inflammation. Although hyperinsulinemia is generally thought to result from an attempt of the ß-cell to compensate for insulin resistance, there is evidence that hyperinsulinaemia itself may contribute to the development of insulin resistance and possibly the low-grade inflammation. To test this hypothesis, U937 macrophages were exposed to insulin. In these cells, insulin induced expression of the proinflammatory cytokines IL-1ß, IL-8, CCL2, and OSM. The insulin-elicited induction of IL-1ß was independent of the presence of endotoxin and most likely mediated by an insulin-dependent activation of NF-κB. Supernatants of the insulin-treated U937 macrophages rendered primary cultures of rat hepatocytes insulin resistant; they attenuated the insulin-dependent induction of glucokinase by 50%. The cytokines contained in the supernatants of insulin-treated U937 macrophages activated ERK1/2 and IKKß, resulting in an inhibitory serine phosphorylation of the insulin receptor substrate. In addition, STAT3 was activated and SOCS3 induced, further contributing to the interruption of the insulin receptor signal chain in hepatocytes. These results indicate that hyperinsulinemia per se might contribute to the low-grade inflammation prevailing in overweight and obese patients and thereby promote the development of insulin resistance particularly in the liver, because the insulin concentration in the portal circulation is much higher than in all other tissues.

Involvement of sphingosine 1-phosphate in palmitate-induced insulin resistance of hepatocytes via the S1P2 receptor subtype.

AIMS/HYPOTHESIS: Enhanced plasma levels of NEFA have been shown to induce hepatic insulin resistance, which contributes to the development of type 2 diabetes. Indeed, sphingolipids can be formed via a de novo pathway from the saturated fatty acid palmitate and the amino acid serine. Besides ceramides, sphingosine 1-phosphate (S1P) has been identified as a major bioactive lipid mediator. Therefore, our aim was to investigate the generation and function of S1P in hepatic insulin resistance. METHODS: The incorporation of palmitate into sphingolipids was performed by rapid-resolution liquid chromatography-MS/MS in primary human and rat hepatocytes. The influence of S1P and the involvement of S1P receptors in hepatic insulin resistance was examined in human and rat hepatocytes, as well as in New Zealand obese (NZO) mice. RESULTS: Palmitate induced an impressive formation of extra- and intracellular S1P in rat and human hepatocytes. An elevation of hepatic S1P levels was observed in NZO mice fed a high-fat diet. Once generated, S1P was able, similarly to palmitate, to counteract insulin signalling. The inhibitory effect of S1P was abolished in the presence of the S1P2 receptor antagonist JTE-013 both in vitro and in vivo. In agreement with this, the immunomodulator FTY720-phosphate, which binds to all S1P receptors except S1P2, was not able to inhibit insulin signalling. CONCLUSIONS/INTERPRETATION: These data indicate that palmitate is metabolised by hepatocytes to S1P, which acts via stimulation of the S1P2 receptor to impair insulin signalling. In particular, S1P2 inhibition could be considered as a novel therapeutic target for the treatment of insulin resistance.

Stimulation of fat accumulation in hepatocytes by PGE2-dependent repression of hepatic lipolysis, ß-oxidation and VLDL-synthesis.

Hepatic steatosis is recognized as hepatic presentation of the metabolic syndrome. Hyperinsulinaemia, which shifts fatty acid oxidation to de novo lipogenesis and lipid storage in the liver, appears to be a principal elicitor particularly in the early stages of disease development. The impact of PGE2, which has previously been shown to attenuate insulin signaling and hence might reduce insulin-dependent lipid accumulation, on insulin-induced steatosis of hepatocytes was studied. The PGE2-generating capacity was enhanced in various obese mouse models by the induction of cyclooxygenase 2 and microsomal prostaglandin E-synthases (mPGES1, mPGES2). PGE2 attenuated the insulin-dependent induction of SREBP-1c and its target genes glucokinase and fatty acid synthase. Nevertheless, PGE2 enhanced incorporation of glucose into hepatic triglycerides synergistically with insulin. This was most likely due to a combination of a PGE2-dependent repression of (1) the key lipolytic enzyme adipose triglyceride lipase, (2) carnitine-palmitoyltransferase 1, a key regulator of mitochondrial ß-oxidation, and (3) microsomal transfer protein, as well as (4) apolipoprotein B, key components of the VLDL synthesis. Repression of PGC1α, a common upstream regulator of these genes, was identified as a possible cause. In support of this hypothesis, overexpression of PGC1α completely blunted the PGE2-dependent fat accumulation. PGE2 enhanced lipid accumulation synergistically with insulin, despite attenuating insulin signaling and might thus contribute to the development of hepatic steatosis. Induction of enzymes involved in PGE2 synthesis in in vivo models of obesity imply a potential role of prostanoids in the development of NAFLD and NASH.

Female mice are more susceptible to nonalcoholic fatty liver disease: sex-specific regulation of the hepatic AMP-activated protein kinase-plasminogen activator inhibitor 1 cascade, but not the hepatic endotoxin response.

As significant differences between sexes were found in the susceptibility to alcoholic liver disease in human and animal models, it was the aim of the present study to investigate whether female mice also are more susceptible to the development of non-alcoholic fatty liver disease (NAFLD). Male and female C57BL/6J mice were fed either water or 30% fructose solution ad libitum for 16 wks. Liver damage was evaluated by histological scoring. Portal endotoxin levels and markers of Kupffer cell activation and insulin resistance, plasminogen activator inhibitor 1 (PAI-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK ) were measured in the liver. Adiponectin mRNA expression was determined in adipose tissue. Hepatic steatosis was almost similar between male and female mice; however, inflammation was markedly more pronounced in livers of female mice. Portal endotoxin levels, hepatic levels of myeloid differentiation primary response gene (88) (MyD88) protein and of 4-hydroxynonenal protein adducts were elevated in animals with NAFLD regardless of sex. Expression of insulin receptor substrate 1 and 2 was decreased to a similar extent in livers of male and female mice with NAFLD. The less pronounced susceptibility to liver damage in male mice was associated with a superinduction of hepatic pAMPK in these mice whereas, in livers of female mice with NAFLD, PAI-1 was markedly induced. Expression of adiponectin in visceral fat was significantly lower in female mice with NAFLD but unchanged in male mice compared with respective controls. In conclusion, our data suggest that the sex-specific differences in the susceptibility to NAFLD are associated with differences in the regulation of the adiponectin-AMPK-PAI-1 signaling cascade.

Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay.

Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.

The Reaction Specificity of Mammalian ALOX15 Orthologs is Changed During Late Primate Evolution and These Alterations Might Offer Evolutionary Advantages for Hominidae.

Arachidonic acid lipoxygenases (ALOXs) have been implicated in the immune response of mammals. The reaction specificity of these enzymes is decisive for their biological functions and ALOX classification is based on this enzyme property. Comparing the amino acid sequences and the functional properties of selected mammalian ALOX15 orthologs we previously hypothesized that the reaction specificity of these enzymes can be predicted based on their amino acid sequences (Triad Concept) and that mammals, which are ranked in evolution below gibbons, express arachidonic acid 12-lipoxygenating ALOX15 orthologs. In contrast, Hominidae involving the great apes and humans possess 15-lipoxygenating enzymes (Evolutionary Hypothesis). These two hypotheses were based on sequence data of some 60 mammalian ALOX15 orthologs and about half of them were functionally characterized. Here, we compared the ALOX15 sequences of 152 mammals representing all major mammalian subclades expressed 44 novel ALOX15 orthologs and performed extensive mutagenesis studies of their triad determinants. We found that ALOX15 genes are absent in extant Prototheria but that corresponding enzymes frequently occur in Metatheria and Eutheria. More than 90% of them catalyze arachidonic acid 12-lipoxygenation and the Triad Concept is applicable to all of them. Mammals ranked in evolution above gibbons express arachidonic acid 15-lipoxygenating ALOX15 orthologs but enzymes with similar specificity are only present in less than 5% of mammals ranked below gibbons. This data suggests that ALOX15 orthologs have been introduced during Prototheria-Metatheria transition and put the Triad Concept and the Evolutionary Hypothesis on a much broader and more reliable experimental basis.

Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging.

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in cell differentiation and in the pathogenesis of inflammation. The mouse genome involves seven functional Alox genes and the encoded enzymes share a high degree of amino acid conservation with their human orthologs. There are, however, functional differences between mouse and human ALOX orthologs. Human ALOX15B oxygenates arachidonic acid exclusively to its 15-hydroperoxy derivative (15S-HpETE), whereas 8S-HpETE is dominantly formed by mouse Alox15b. The structural basis for this functional difference has been explored and in vitro mutagenesis humanized the reaction specificity of the mouse enzyme. To explore whether this mutagenesis strategy may also humanize the reaction specificity of mouse Alox15b in vivo, we created Alox15b knock-in mice expressing the arachidonic acid 15-lipoxygenating Tyr603Asp+His604Val double mutant instead of the 8-lipoxygenating wildtype enzyme. These mice are fertile, display slightly modified plasma oxylipidomes and develop normally up to an age of 24 weeks. At later developmental stages, male Alox15b-KI mice gain significantly less body weight than outbred wildtype controls, but this effect was not observed for female individuals. To explore the possible reasons for the observed gender-specific growth arrest, we determined the basic hematological parameters and found that aged male Alox15b-KI mice exhibited significantly attenuated red blood cell parameters (erythrocyte counts, hematocrit, hemoglobin). Here again, these differences were not observed in female individuals. These data suggest that humanization of the reaction specificity of mouse Alox15b impairs the functionality of the hematopoietic system in males, which is paralleled by a premature growth arrest.

Oncostatin M produced in Kupffer cells in response to PGE2: possible contributor to hepatic insulin resistance and steatosis.

Hepatic insulin resistance is a major contributor to hyperglycemia in metabolic syndrome and type II diabetes. It is caused in part by the low-grade inflammation that accompanies both diseases, leading to elevated local and circulating levels of cytokines and cyclooxygenase (COX) products such as prostaglandin E(2) (PGE(2)). In a recent study, PGE(2) produced in Kupffer cells attenuated insulin-dependent glucose utilization by interrupting the intracellular signal chain downstream of the insulin receptor in hepatocytes. In addition to directly affecting insulin signaling in hepatocytes, PGE(2) in the liver might affect insulin resistance by modulating cytokine production in non-parenchymal cells. In accordance with this hypothesis, PGE(2) stimulated oncostatin M (OSM) production by Kupffer cells. OSM in turn attenuated insulin-dependent Akt activation and, as a downstream target, glucokinase induction in hepatocytes, most likely by inducing suppressor of cytokine signaling 3 (SOCS3). In addition, it inhibited the expression of key enzymes of hepatic lipid metabolism. COX-2 and OSM mRNA were induced early in the course of the development of non-alcoholic steatohepatitis (NASH) in mice. Thus, induction of OSM production in Kupffer cells by an autocrine PGE(2)-dependent feed-forward loop may be an additional, thus far unrecognized, mechanism contributing to hepatic insulin resistance and the development of NASH.

Cell-Based Reporter Release Assay to Determine the Activity of Calcium-Dependent Neurotoxins and Neuroactive Pharmaceuticals.

The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.

Aggravation by prostaglandin E2 of interleukin-6-dependent insulin resistance in hepatocytes.

UNLABELLED: Hepatic insulin resistance is a major contributor to fasting hyperglycemia in patients with metabolic syndrome and type 2 diabetes. Circumstantial evidence suggests that cyclooxygenase products in addition to cytokines might contribute to insulin resistance. However, direct evidence for a role of prostaglandins in the development of hepatic insulin resistance is lacking. Therefore, the impact of prostaglandin E(2) (PGE(2)) alone and in combination with interleukin-6 (IL-6) on insulin signaling was studied in primary hepatocyte cultures. Rat hepatocytes were incubated with IL-6 and/or PGE(2) and subsequently with insulin. Glycogen synthesis was monitored by radiochemical analysis; the activation state of proteins of the insulin receptor signal chain was analyzed by western blot with phosphospecific antibodies. In hepatocytes, insulin-stimulated glycogen synthesis and insulin-dependent phosphorylation of Akt-kinase were attenuated synergistically by prior incubation with IL-6 and/or PGE(2) while insulin receptor autophosphorylation was barely affected. IL-6 but not PGE(2) induced suppressors of cytokine signaling (SOCS3). PGE(2) but not IL-6 activated extracellular signal-regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE(2)-dependent but not the IL-6-dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3-receptor, PGE(2) pre-incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin-dependent Akt-phosphorylation. CONCLUSION: PGE(2) might contribute to hepatic insulin resistance via an EP3-receptor-dependent ERK1/2 activation resulting in a serine phosphorylation of insulin receptor substrate, thereby preventing an insulin-dependent activation of Akt and glycogen synthesis. Since different molecular mechanisms appear to be employed, PGE(2) may synergize with IL-6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3.

Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays.

Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.

Reduced Oxidative Stress and Enhanced FGF21 Formation in Livers of Endurance-Exercised Rats with Diet-Induced NASH.

Non-alcoholic fatty liver diseases (NAFLD) including the severe form with steatohepatitis (NASH) are highly prevalent ailments to which no approved pharmacological treatment exists. Dietary intervention aiming at 10% weight reduction is efficient but fails due to low compliance. Increase in physical activity is an alternative that improved NAFLD even in the absence of weight reduction. The underlying mechanisms are unclear and cannot be studied in humans. Here, a rat NAFLD model was developed that reproduces many facets of the diet-induced NAFLD in humans. The impact of endurance exercise was studied in this model. Male Wistar rats received control chow or a NASH-inducing diet rich in fat, cholesterol, and fructose. Both diet groups were subdivided into a sedentary and an endurance exercise group. Animals receiving the NASH-inducing diet gained more body weight, got glucose intolerant and developed a liver pathology with steatosis, hepatocyte hypertrophy, inflammation and fibrosis typical of NAFLD or NASH. Contrary to expectations, endurance exercise did not improve the NASH activity score and even enhanced hepatic inflammation. However, endurance exercise attenuated the hepatic cholesterol overload and the ensuing severe oxidative stress. In addition, exercise improved glucose tolerance possibly in part by induction of hepatic FGF21 production.

Dietary cholesterol does not break your heart but kills your liver.

It is increasingly accepted that dietary cholesterol has a much lower impact on the progression of cardiovascular disease than previously assumed. However, both animal experiments and human studies seem to support the view that dietary cholesterol may contribute to the transition from benign steatosis to the potentially fatal non-alcoholic steatohepatitis. Cholesterol esters and cholesterol accumulate in the hepatocyte and impair its function. This leads to oxidative stress and endoplasmic reticulum stress triggering the release of pro-inflammatory cytokines and rendering the hepatocyte more susceptible to apoptotic or necrotic cell death. Kupffer cells group around dying hepatocytes and phagocytose the hepatocyte debris and lipids. In addition, they are exposed to lipid peroxidation products released from hepatocytes. Kupffer cells, thus activated, release pro-inflammatory, chemotactic and profibrotic cytokines that promote inflammation and fibrosis. Therefore, dietary cholesterol may be harmful to the liver, in particular when administered in combination with polyunsaturated fatty acids that favor lipid peroxidation.

Soybean Oil-Derived Poly-Unsaturated Fatty Acids Enhance Liver Damage in NAFLD Induced by Dietary Cholesterol.

While the impact of dietary cholesterol on the progression of atherosclerosis has probably been overestimated, increasing evidence suggests that dietary cholesterol might favor the transition from blunt steatosis to non-alcoholic steatohepatitis (NASH), especially in combination with high fat diets. It is poorly understood how cholesterol alone or in combination with other dietary lipid components contributes to the development of lipotoxicity. The current study demonstrated that liver damage caused by dietary cholesterol in mice was strongly enhanced by a high fat diet containing soybean oil-derived ω6-poly-unsaturated fatty acids (ω6-PUFA), but not by a lard-based high fat diet containing mainly saturated fatty acids. In contrast to the lard-based diet the soybean oil-based diet augmented cholesterol accumulation in hepatocytes, presumably by impairing cholesterol-eliminating pathways. The soybean oil-based diet enhanced cholesterol-induced mitochondrial damage and amplified the ensuing oxidative stress, probably by peroxidation of poly-unsaturated fatty acids. This resulted in hepatocyte death, recruitment of inflammatory cells, and fibrosis, and caused a transition from steatosis to NASH, doubling the NASH activity score. Thus, the recommendation to reduce cholesterol intake, in particular in diets rich in ω6-PUFA, although not necessary to reduce the risk of atherosclerosis, might be sensible for patients suffering from non-alcoholic fatty liver disease.

Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells.

Analyzing mixture toxicity requires an in-depth understanding of the mechanisms of action of its individual components. Substances with the same target organ, same toxic effect and same mode of action (MoA) are believed to cause additive effects, whereas substances with different MoAs are assumed to act independently. Here, we tested 2 triazole fungicides, propiconazole, and tebuconazole (Te), for individual and combined effects on liver toxicity-related endpoints. Both triazoles are proposed to belong to the same cumulative assessment group and are therefore thought to display similar and additive behavior. Our data show that Te is an antagonist of the constitutive androstane receptor (CAR) in rats and humans, while propiconazole is an agonist of this receptor. Both substances activate the pregnane X-receptor (PXR) and further induce mRNA expression of CYP3A4. CYP3A4 enzyme activity, however, is inhibited by propiconazole. For common targets of PXR and CAR, the activation of PXR by Te overrides CAR inhibition. In summary, propiconazole and Te affect different hepatotoxicity-relevant cellular targets and, depending on the individual endpoint analyzed, act via similar or dissimilar mechanisms. The use of molecular data based on research in human cell systems extends the picture to refine cumulative assessment group grouping and substantially contributes to the understanding of mixture effects of chemicals in biological systems.

PPARalpha-dependent induction of the energy homeostasis-regulating nuclear receptor NR1i3 (CAR) in rat hepatocytes: potential role in starvation adaptation.

A tight hormonal control of energy homeostasis is of pivotal relevance for animals. Recent evidence suggests an involvement of the nuclear receptor NR1i3 (CAR). Fasting induces CAR by largely unknown mechanisms and CAR-deficient mice are defective in fasting adaptation. In rat hepatocytes CAR was induced by WY14643, a PPARalpha-agonist. A DR1 motif in the CAR promoter was necessary and sufficient for this control. The PPARalpha-dependent increase in CAR potentiated the phenobarbital-induced transcription of the prototypical CAR-dependent gene CYP2B1. Since free fatty acids are natural ligands for PPARalpha, a fasting-induced increase in free fatty acids might induce CAR. In accordance with this hypothesis, CAR induction by fasting was abrogated in PPARalpha-deficient mice.

Role of the ERC motif in the proximal part of the second intracellular loop and the C-terminal domain of the human prostaglandin F2alpha receptor (hFP-R) in G-protein coupling control.

The human FP-R (F2alpha prostaglandin receptor) is a Gq-coupled heptahelical ectoreceptor, which is of significant medical interest, since it is a potential target for the treatment of glaucoma and preterm labour. On agonist exposure, it mediates an increase in intracellular inositol phosphate formation. Little is known about the structures that govern the agonist-dependent receptor activation. In other prostanoid receptors, the C-terminal domain has been inferred in the control of agonist-dependent receptor activation. A DRY motif at the beginning of the second intracellular loop is highly conserved throughout the G-protein-coupled receptor family and appears to be crucial for controlling agonist-dependent receptor activation. It is replaced by an ERC motif in the FP-R and no evidence for the relevance of this motif in ligand-dependent activation of prostanoid receptors has been provided so far. The aim of the present study was to elucidate the potential role of the C-terminal domain and the ERC motif in agonist-controlled intracellular signalling in FP-R mutants generated by site-directed mutagenesis. It was found that substitution of the acidic Glu(132) in the ERC motif by a threonine residue led to full constitutive activation, whereas truncation of the receptor's C-terminal domain led to partial constitutive activation of all three intracellular signal pathways that had previously been shown to be activated by the FP-R, i.e. inositol trisphosphate formation, focal adhesion kinase activation and T-cell factor signalling. Inositol trisphosphate formation and focal adhesion kinase phosphorylation were further enhanced by ligand binding in cells expressing the truncation mutant but not the E132T (Glu132-->Thr) mutant. Thus C-terminal truncation appeared to result in a receptor with partial constitutive activation, whereas substitution of Glu132 by threonine apparently resulted in a receptor with full constitutive activity.