TY - GEN A1 - Michaud Schjeide, Brit-Maren A1 - Schenke, Maren A1 - Seeger, Bettina A1 - Püschel, Gerhard Paul T1 - Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1269 KW - CRISPR editing validation KW - copy number analyses KW - homology-directed repair KW - homologous recombination deficiency Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-561755 SN - 1866-8372 SP - 1 EP - 14 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - JOUR A1 - Schjeide, Brit-Maren A1 - Schenke, Maren A1 - Seeger, Bettina A1 - Püschel, Gerhard T1 - Validation of a novel double control quantitative copy number PCR method to quantify off-target transgene integration after CRISPR-induced DNA modification JF - Methods and protocols : M&Ps N2 - In order to improve a recently established cell-based assay to assess the potency of botulinum neurotoxin, neuroblastoma-derived SiMa cells and induced pluripotent stem-cells (iPSC) were modified to incorporate the coding sequence of a reporter luciferase into a genetic safe harbor utilizing CRISPR/Cas9. A novel method, the double-control quantitative copy number PCR (dc-qcnPCR), was developed to detect off-target integrations of donor DNA. The donor DNA insertion success rate and targeted insertion success rate were analyzed in clones of each cell type. The dc-qcnPCR reliably quantified the copy number in both cell lines. The probability of incorrect donor DNA integration was significantly increased in SiMa cells in comparison to the iPSCs. This can possibly be explained by the lower bundled relative gene expression of a number of double-strand repair genes (BRCA1, DNA2, EXO1, MCPH1, MRE11, and RAD51) in SiMa clones than in iPSC clones. The dc-qcnPCR offers an efficient and cost-effective method to detect off-target CRISPR/Cas9-induced donor DNA integrations. KW - CRISPR editing validation KW - copy number analyses KW - homology-directed repair KW - homologous recombination deficiency Y1 - 2022 U6 - https://doi.org/10.3390/mps5030043 SN - 2409-9279 VL - 5 IS - 3 SP - 1 EP - 14 PB - MDPI CY - Basel, Schweiz ER - TY - JOUR A1 - Knebel, Constanze A1 - Neeb, Jannika A1 - Zahn, Elisabeth A1 - Schmidt, Flavia A1 - Carazo, Alejandro A1 - Holas, Ondej A1 - Pavek, Petr A1 - Püschel, Gerhard Paul A1 - Zanger, Ulrich M. A1 - Süssmuth, Roderich A1 - Lampen, Alfonso A1 - Marx-Stoelting, Philip A1 - Braeuning, Albert T1 - Unexpected Effects of Propiconazole, Tebuconazole, and Their Mixture on the Receptors CAR and PXR in Human Liver Cells JF - Toxicological sciences N2 - 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. KW - triazole fungicides KW - constitutive androstane receptor KW - pregnane X-receptor KW - enzyme induction KW - liver toxicity KW - mixtures Y1 - 2018 U6 - https://doi.org/10.1093/toxsci/kfy026 SN - 1096-6080 SN - 1096-0929 VL - 163 IS - 1 SP - 170 EP - 181 PB - Oxford Univ. Press CY - Oxford ER - TY - CHAP A1 - Henkel, Janine A1 - Camargo, Rodolfo Gonzalez A1 - Schanze, Nancy A1 - Püschel, Gerhard Paul T1 - The vicious circle of prostaglandin- and cytokine-dependent hepatic insulin resistance: a key role of prostaglandin E2 T2 - Diabetologia : journal of the European Association for the Study of Diabetes (EASD) Y1 - 2014 SN - 0012-186X SN - 1432-0428 VL - 57 SP - S241 EP - S242 PB - Springer CY - New York ER - TY - JOUR A1 - Neuschaefer-Rube, Frank A1 - Lieske, Stefanie A1 - Kuna, Manuela A1 - Henkel, Janin A1 - Perry, Rachel J. A1 - Erion, Derek M. A1 - Pesta, Dominik A1 - Willmes, Diana M. A1 - Brachs, Sebastian A1 - von Loeffelholz, Christian A1 - Tolkachov, Alexander A1 - Schupp, Michael A1 - Pathe-Neuschaefer-Rube, Andrea A1 - Pfeiffer, Andreas F. H. A1 - Shulman, Gerald I. A1 - Püschel, Gerhard Paul A1 - Birkenfeld, Andreas L. T1 - The mammalian INDY homolog is induced by CREB in a rat model of type 2 diabetes JF - Diabetes : a journal of the American Diabetes Association Y1 - 2014 SN - 0012-1797 SN - 1939-327X VL - 63 IS - 3 SP - 1048 EP - 1057 PB - American Diabetes Association CY - Alexandria ER - TY - JOUR A1 - von Loeffelholz, Christian A1 - Lieske, Stefanie A1 - Neuschaefer-Rube, Frank A1 - Willmes, Diana M. A1 - Raschzok, Nathanael A1 - Sauer, Igor M. A1 - König, Jörg A1 - Fromm, Martin F. A1 - Horn, Paul A1 - Chatzigeorgiou, Antonios A1 - Pathe-Neuschaefer-Rube, Andrea A1 - Jordan, Jens A1 - Pfeiffer, Andreas F. H. A1 - Mingrone, Geltrude A1 - Bornstein, Stefan R. A1 - Stroehle, Peter A1 - Harms, Christoph A1 - Wunderlich, F. Thomas A1 - Helfand, Stephen L. A1 - Bernier, Michel A1 - de Cabo, Rafael A1 - Shulman, Gerald I. A1 - Chavakis, Triantafyllos A1 - Püschel, Gerhard Paul A1 - Birkenfeld, Andreas L. T1 - The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism BT - official journal of the American Association for the Study of Liver Diseases JF - Hepatology N2 - 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. Y1 - 2017 U6 - https://doi.org/10.1002/hep.29089 SN - 0270-9139 SN - 1527-3350 VL - 66 IS - 2 SP - 616 EP - 630 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Wieneke, Nadine A1 - Neuschaefer-Rube, Frank A1 - Bode, L. M. A1 - Kuna, Manuela A1 - Andres, Jesus A1 - Carnevali Junior, Luiz Carlos A1 - Hirsch-Ernst, Karen I. A1 - Püschel, Gerhard Paul T1 - Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes N2 - Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting- incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/0041008X U6 - https://doi.org/10.1016/j.taap.2009.07.014 SN - 0041-008X ER - TY - JOUR A1 - Neuschäfer-Rube, Frank A1 - Möller, Ulrike A1 - Püschel, Gerhard Paul T1 - Structure of the 5'-flanking region of the rat prostaglandin f(2alpha) receptor N2 - Prostaglandin F(2alpha) (PGF(2alpha)), modulates hepatocyte functions via a heptahelical G(q)-coupled PGF(2alpha)-receptor (FP-R) which in liver is expressed exclusively in hepatocytes. The aim of the present study was to isolate the 5'-flanking region of the rat FP-R gene and to elucidate its basal and IL-6-modulated transcription control function in rat hepatocytes. The 5'-non-translated region of the rat hepatocyte FP-R mRNA differed from the corresponding region in rat fetal astrocyte or corpus luteum. It was encoded by exons 1a and 2 which were separated by a 1. 4 kb intron containing the exons 1b and 1c coding for the 5'-untranslated region of rat fetal astrocyte and corpus luteum FP-R mRNA, respectively. The transcription initiation site in hepatocytes was localized 263 bp upstream of the start ATG by 5'-RACE. A DNA-fragment covering the 5'-flanking region of the rFP-R gene from - 1 of the transcription initiation site to -2590 bp was cloned and sequenced. Its 3'-two thirds had a 65% sequence identity to the mouse FP-R promoter however no homology to the bovine FP-R promoter. In the overlapping sequence most of the putative transcription factor binding sites were conserved between mouse and rat. The rat promoter contained no classical TATA- or CAAT-boxes but putative binding sites for the transcription factors C/EBP, GATA-1, HNF-1, HNF-3beta, SP-1, and USF. Luciferase reporter gene constructs containing portions of the 5'-flanking region were transfected into rat hepatocytes. Luciferase expression ranked -181 >/= -608 < -1418 > -1821 >/= -2590. The strongest transcriptional activity was conferred by the region between -608 and -1418 containing a cluster of potential HNF-1 and HNF-3beta binding sites that might allow the exclusive expression of FP-R mRNA in hepatocytes. The amount of FP-R mRNA and the luciferase expression under control of the -2590 promoter fragment were reduced by IL-6 in hepatocytes. Copyright 2000 Academic Press. Y1 - 2000 ER - TY - GEN A1 - Hespeling, Ursula A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt A1 - Götze, Otto A1 - Zwirner, Jörg T1 - Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co-cultures N2 - Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 pg/ml increased the formation of thromboxane A₂, prostaglandin D₂, E₂ and F₂α6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 μM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 117 Y1 - 1995 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45909 ER - TY - JOUR A1 - Henkel, Janin A1 - Frede, Katja A1 - Schanze, Nancy A1 - Vogel, Heike A1 - Schürmann, Annette A1 - Spruß, Astrid A1 - Bergheim, Ina A1 - Püschel, Gerhard Paul T1 - Stimulation of fat accumulation in hepatocytes by PGE(2)-dependent repression of hepatic lipolysis, beta-oxidation and VLDL-synthesis JF - Laboratory investigation : the basic and translational pathology research journal ; an official journal of the United States and Canadian Academy of Pathology N2 - 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 PGE(2), 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 PGE(2)-generating capacity was enhanced in various obese mouse models by the induction of cyclooxygenase 2 and microsomal prostaglandin E-synthases (mPGES1, mPGES2). PGE(2) attenuated the insulin-dependent induction of SREBP-1c and its target genes glucokinase and fatty acid synthase. Nevertheless, PGE(2) enhanced incorporation of glucose into hepatic triglycerides synergistically with insulin. This was most likely due to a combination of a PGE(2)-dependent repression of (1) the key lipolytic enzyme adipose triglyceride lipase, (2) carnitine-palmitoyltransferase 1, a key regulator of mitochondrial beta-oxidation, and (3) microsomal transfer protein, as well as (4) apolipoprotein B, key components of the VLDL synthesis. Repression of PGC1 alpha, a common upstream regulator of these genes, was identified as a possible cause. In support of this hypothesis, overexpression of PGC1 alpha completely blunted the PGE(2)-dependent fat accumulation. PGE(2) 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 PGE(2) synthesis in in vivo models of obesity imply a potential role of prostanoids in the development of NAFLD and NASH. Laboratory Investigation (2012) 92, 1597-1606; doi:10.1038/labinvest.2012.128; published online 10 September 2012 KW - cyclooxygenase KW - hepatic steatosis KW - mPGES KW - NAFLD KW - NASH KW - type 2 diabetes (T2DM) KW - PGC1 alpha Y1 - 2012 U6 - https://doi.org/10.1038/labinvest.2012.128 SN - 0023-6837 VL - 92 IS - 11 SP - 1597 EP - 1606 PB - Nature Publ. Group CY - New York ER - TY - JOUR A1 - Henkel, Janin A1 - Alfine, Eugenia A1 - Saín, Juliana A1 - Jöhrens, Korinna A1 - Weber, Daniela A1 - Castro, José Pedro A1 - König, Jeannette A1 - Stuhlmann, Christin A1 - Vahrenbrink, Madita A1 - Jonas, Wenke A1 - Kleinridders, André A1 - Püschel, Gerhard Paul T1 - Soybean Oil-Derived Poly-Unsaturated Fatty Acids Enhance Liver Damage in NAFLD Induced by Dietary Cholesterol JF - Nutrients N2 - 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. KW - non-alcoholic fatty liver disease (NAFLD) KW - NASH KW - cholesterol KW - PUFA KW - inflammation KW - oxidative stress Y1 - 2018 U6 - https://doi.org/10.3390/nu10091326 SN - 2072-6643 VL - 10 IS - 9 SP - 1 EP - 17 PB - Molecular Diversity Preservation International (MDPI) CY - Basel ER - TY - GEN A1 - Henkel, Janin A1 - Alfine, Eugenia A1 - Saín, Juliana A1 - Jöhrens, Korinna A1 - Weber, Daniela A1 - Castro, José Pedro A1 - König, Jeannette A1 - Stuhlmann, Christin A1 - Vahrenbrink, Madita A1 - Jonas, Wenke A1 - Kleinridders, André A1 - Püschel, Gerhard Paul T1 - Soybean Oil-Derived Poly-Unsaturated Fatty Acids Enhance Liver Damage in NAFLD Induced by Dietary Cholesterol T2 - Nutrients N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 479 KW - non-alcoholic fatty liver disease (NAFLD) KW - NASH KW - cholesterol KW - PUFA KW - inflammation KW - oxidative stress Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-419773 ER - TY - CHAP A1 - Schenke, Maren A1 - Schjeide, Brit-Maren A1 - Püschel, Gerhard Paul A1 - Seeger, Bettina T1 - Serotype-specific sensitivity to Botulinum neurotoxins of iPSC-derived motor neurons T2 - Naunyn-Schmiedeberg's archives of pharmacology Y1 - 2021 U6 - https://doi.org/10.1007/s00210-021-02066-6 SN - 0028-1298 SN - 1432-1912 VL - 394 IS - Suppl. 1 SP - S4 EP - S4 PB - Springer CY - Berlin ; Heidelberg ER - TY - JOUR A1 - Böer, Ulrike A1 - Fennekohl, Alexandra A1 - Püschel, Gerhard Paul T1 - Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor alpha-induced apoptosis N2 - BACKGROUND/AIMS: Tumor necrosis factor (TNF) elicits hepatocyte apoptosis in toxic liver injury and is also central in hepatocyte proliferation after partial hepatectomy. In both circumstances interleukin (IL)-6 levels are also elevated. In mouse liver IL-6 attenuated Fas receptor-mediated apoptosis indicating its interference with pro-apoptotic signal chains. It was, therefore, the aim to examine the modulation by IL-6 of TNFalpha-induced apoptosis in rat hepatocytes. METHODS: Primary rat hepatocytes were treated with IL-6 prior to induction of apoptosis with TNFalpha/ actinomycin D or anti-Fas antibody M-20. Apoptosis was detected by determination of caspase-3 activation and bisbenzimide staining of condensed nuclei. Expression of TNFalpha receptors was analyzed by semi-quantitative polymerase chain reaction and ligand binding studies with [125I]-TNFalpha. RESULTS: IL-6 treatment doubled TNFalpha/actinomycin D- induced caspase-3 activity and significantly enhanced chromatin condensation. By contrast IL-6 inhibited Fas-induced increase in caspase-3 activity by 45% and significantly reduced chromatin condensation. IL-6 increased the mRNA level of TNF-R1 1.35-fold and augmented cell surface binding of [125I]-TNFalpha 3-fold. The latter and TNFalpha-mediated caspase activation was attenuated by prostaglandin E(2). CONCLUSIONS: IL-6 - in contrast to its anti-apoptotic modulation of the Fas-induced pathway - exerted a pro-apoptotic effect on the TNFalpha/actinomycin D-induced apoptosis by increasing the number of TNF-R on hepatocytes. Y1 - 2003 UR - http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=12763364 ER - TY - JOUR A1 - Pathe-Neuschäfer-Rube, Andrea A1 - Neuschäfer-Rube, Frank A1 - Püschel, Gerhard Paul T1 - 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 N2 - 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. Y1 - 2005 UR - http://www.biochemj.org/bj/388/0317/bj3880317.htm ER - TY - JOUR A1 - Böer, Ulrike A1 - Neuschäfer-Rube, Frank A1 - Möller, Ulrike A1 - Püschel, Gerhard Paul T1 - Requirement of N-glycosylation of the prostaglandin E2 receptor EP3beta for correct sorting to the plasma membrane but not for correct folding N2 - Eight heptahelical receptors have been characterized for prostaglandin (PG) D(2), PGE(2), PGF(2alpha), prostacyclin and thromboxane A(2). They share a sequence identity of 40%. All of them have potential N-glycosylation sites. The current study analysed the role of the two N-glycosylation sites in the rat EP3beta-subtype PGE(2) receptor for protein folding and sorting. The N-glycosylation consensus sequences were eliminated by site-directed mutagenesis and receptors expressed in HEK-293 cells. Both potential N-glycosylation sites were used. Their joint elimination resulted in the synthesis of a receptor protein with full binding competence, biological activity and no reduction of affinity; however, the half-life of the non-glycosylated receptor was slightly reduced. Ligand binding to intact stably transfected cells and confocal laser microscopic immunocytochemistry showed that the glycosylated receptor was correctly inserted into the plasma membrane to a much larger extent than the non-glycosylated receptor, which tended to accumulate in the perinuclear zone of the endoplasmic reticulum. Inhibition of N-glycosylation with tunicamycin resulted in a similar perinuclear distribution of the wild-type receptor. Therefore, glycosylation of the EP3beta receptor seems not to be necessary for correct folding of the receptor protein but for the efficient transport of the receptor protein to the plasma membrane. This contrasts with a previous finding which described a reduction of the affinity for PGE(2) of the EP3alpha receptor by elimination of the distal glycosylation site when the receptor protein was expressed in insect cells. Y1 - 2000 ER - TY - GEN A1 - Henkel, Janin A1 - Buchheim-Dieckow, Katja A1 - Castro, José Pedro A1 - Laeger, Thomas A1 - Wardelmann, Kristina A1 - Kleinridders, André A1 - Jöhrens, Korinna A1 - Püschel, Gerhard Paul T1 - Reduced Oxidative Stress and Enhanced FGF21 Formation in Livers of Endurance-Exercised Rats with Diet-Induced NASH T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 807 KW - NAFLD KW - NASH KW - endurance exercise KW - FGF21 KW - glucose intolerance KW - cholesterol KW - oxidative stress Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-442384 SN - 1866-8372 IS - 807 ER - TY - JOUR A1 - Henkel, Janin A1 - Buchheim-Dieckow, Katja A1 - Castro, José Pedro A1 - Laeger, Thomas A1 - Wardelmann, Kristina A1 - Kleinridders, André A1 - Jöhrens, Korinna A1 - Püschel, Gerhard Paul T1 - Reduced Oxidative Stress and Enhanced FGF21 Formation in Livers of Endurance-Exercised Rats with Diet-Induced NASH JF - Nutrients N2 - 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. KW - NAFLD KW - NASH KW - endurance exercise KW - FGF21 KW - glucose intolerance KW - cholesterol KW - oxidative stress Y1 - 2019 U6 - https://doi.org/10.3390/nu11112709 SN - 2072-6643 VL - 11 IS - 11 PB - MDPI CY - Basel ER - TY - GEN A1 - Uhlig, Katja A1 - Gehre, Christian P. A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner A1 - Coleman, Charles Dominic A1 - Püschel, Gerhard Paul A1 - Duschl, Claus T1 - Real-time monitoring of oxygen consumption of hepatocytes in a microbioreactor T2 - Toxicology letters Y1 - 2018 U6 - https://doi.org/10.1016/j.toxlet.2018.06.652 SN - 0378-4274 SN - 1879-3169 VL - 295 SP - S115 EP - S115 PB - Elsevier CY - Clare ER - TY - JOUR A1 - Gehre, Christian A1 - Flechner, Marie A1 - Kammerer, Sarah A1 - Küpper, Jan-Heiner A1 - Coleman, Charles Dominic A1 - Püschel, Gerhard Paul A1 - Uhlig, Katja A1 - Duschl, Claus T1 - Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors JF - Scientific reports N2 - Most in vitro test systems for the assessment of toxicity are based on endpoint measurements and cannot contribute much to the establishment of mechanistic models, which are crucially important for further progress in this field. Hence, in recent years, much effort has been put into the development of methods that generate kinetic data. Real time measurements of the metabolic activity of cells based on the use of oxygen sensitive microsensor beads have been shown to provide access to the mode of action of compounds in hepatocytes. However, for fully exploiting this approach a detailed knowledge of the microenvironment of the cells is required. In this work, we investigate the cellular behaviour of three types of hepatocytes, HepG2 cells, HepG2-3A4 cells and primary mouse hepatocytes, towards their exposure to acetaminophen when the availability of oxygen for the cell is systematically varied. We show that the relative emergence of two modes of action, one NAPQI dependent and the other one transient and NAPQI independent, scale with expression level of CYP3A4. The transient cellular response associated to mitochondrial respiration is used to characterise the influence of the initial oxygen concentration in the wells before exposure to acetaminophen on the cell behaviour. A simple model is presented to describe the behaviour of the cells in this scenario. It demonstrates the level of control over the role of oxygen supply in these experiments. This is crucial for establishing this approach into a reliable and powerful method for the assessment of toxicity. Y1 - 2020 U6 - https://doi.org/10.1038/s41598-020-70785-6 SN - 2045-2322 VL - 10 IS - 1 PB - Macmillan Publishers Limited, part of Springer Nature CY - [London] ER - TY - GEN A1 - Watanabe, Yuji A1 - Püschel, Gerhard Paul A1 - Gardemann, Andreas A1 - Jungermann, Kurt T1 - Presinusoidal and proximal intrasinusoidal confluence of hepatic artery and portal vein in rat liver : functional evidence by orthograde and retrograde bivascular perfusion N2 - The site of confluence of the artery and the portal vein in the liver still appears to be controversial. Anatomical studies suggested a presinusoidal or an intrasinusoidal confluence in the first, second or even final third of the sinusoids. The objective of this investigation was to study the problem with functional biochemical techniques. Rat livers were perfused through the hepatic artery and simultaneously either in the orthograde direction from the portal vein to the hepatic vein or in the retrograde direction from the hepatic vein to the portal vein. Arterial how was linearly dependent on arterial pressure between 70 cm H2O and 120 cm H2O at a constant portal or hepatovenous pressure of 18 cm H2O. An arterial pressure of 100 cm H2O was required for the maintenance of a homogeneous orthograde perfusion of the whole parenchyma and of a physiologic ratio of arterial to portal how of about 1:3. Glucagon was infused either through the artery or the portal vein and hepatic vein, respectively, to a submaximally effective ''calculated'' sinusoidal concentration after mixing of 0.1 nmol/L. During orthograde perfusions, arterial and portal glucagon caused the same increases in glucose output. Yet during retrograde perfusions, hepatovenous glucagon elicited metabolic alterations equal to those in orthograde perfusions, whereas arterial glucagon effected changes strongly reduced to between 10% and 50%. Arterially infused trypan blue was distributed homogeneously in the parenchyma during orthograde perfusions, whereas it reached clearly smaller areas of parenchyma during retrograde perfusions. Finally, arterially applied acridine orange was taken up by all periportal hepatocytes in the proximal half of the acinus during orthograde perfusions but only by a much smaller portion of periportal cells in the proximal third of the acinus during retrograde perfusions. These findings suggest that in rat liver, the hepatic artery and the portal vein mix before and within the first third of the sinusoids, rather than in the middle or even last third. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 067 KW - Hepatic artery KW - Portal vein KW - Confluence KW - Rat KW - Animal KW - Experimental study KW - Anatomy KW - Biochemical analysis KW - Technique KW - Perfusion KW - Exploration Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16702 ER - TY - JOUR A1 - Wieneke, Nadine A1 - Hirsch-Ernst, Karen I. A1 - Kuna, Manuela A1 - Kersten, Sander A1 - Püschel, Gerhard Paul T1 - PPARalpha-dependent induction of the energy homeostasis-regulating nuclear N2 - 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. Y1 - 2007 UR - http://www.sciencedirect.com/science/article/pii/S0014579307011556 SN - 0014-5793 ER - TY - JOUR A1 - Rehwald, Matthias A1 - Neuschäfer-Rube, Frank A1 - DeVries, Christa A1 - Püschel, Gerhard Paul T1 - Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2alpha receptor N2 - For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor. Y1 - 1999 ER - TY - JOUR A1 - Neuschäfer-Rube, Frank A1 - Pathe-Neuschäfer-Rube, Andrea A1 - Hippenstiel, Stefan A1 - Püschel, Gerhard Paul T1 - PGE(2) enhanced TNF alpha-mediated IL-8 induction in monocytic cell lines and PBMC JF - Cytokine N2 - Background & purpose: Recent studies suggested a role of prostaglandin E-2 (PGE(2)) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNF alpha-induced IL-8 expression was studied in monocytic cell lines. Experimental approach: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. Key results: In monocytic cell lines THP-1, MonoMac and U937 PGE(2) had only a marginal impact on IL-8 induction but strongly enhanced TNFa-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNF alpha and PGE(2) than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNF alpha-induced IL-8 mRNA and protein formation to the same extent as PGE(2). In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE(2) enhanced TNFainduced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNF alpha-mediated IL-8 mRNA induction by PGE(2) was mimicked by a PICA-activator. Furthermore in these cells PGE(2) induced expression of transcription factor C/EBPS, enhanced NF-KB activation by TNFa and inhibited TNF alpha-mediated AP-1 activation. PGE(2) and TNF alpha synergistically activated transcription factor CREB, induced C/EBPS expression and enhanced the activity of an IL-8 promoter fragment containing-223 bp upstream of the transcription start site. Conclusions and implications: These findings suggest that a combined stimulation of TNF alpha and PGE(2)/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PICA/CREB/C/EB1313 as well as NF-kappa B signal chains. KW - Monocyte KW - Prostaglandin receptor EP4 KW - IL-8 transcription KW - Signal transduction KW - Tumor necrosis factor alpha Y1 - 2018 U6 - https://doi.org/10.1016/j.cyto.2018.06.020 SN - 1043-4666 SN - 1096-0023 VL - 113 SP - 105 EP - 116 PB - Elsevier CY - London ER - TY - JOUR A1 - Henkel, Janin A1 - Gärtner, Daniela A1 - Dorn, Christoph A1 - Hellerbrand, Claus A1 - Schanze, Nancy A1 - Elz, Sheila R. A1 - Püschel, Gerhard Paul T1 - Oncostatin M produced in Kupffer cells in response to PGE(2) possible contributor to hepatic insulin resistance and steatosis JF - Laboratory investigation : the basic and translational pathology research journal ; an official journal of the United States and Canadian Academy of Pathology N2 - 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. KW - cyclooxygenase KW - cytokine KW - hepatic steatosis KW - NASH KW - suppressor of cytokine signaling (SOCS) KW - type II diabetes (T2DM) Y1 - 2011 U6 - https://doi.org/10.1038/labinvest.2011.47 SN - 0023-6837 VL - 91 IS - 7 SP - 1107 EP - 1117 PB - Nature Publ. Group CY - New York ER - TY - JOUR A1 - Neuschäfer-Rube, Frank A1 - Pathe-Neuschäfer-Rube, A. A1 - Hippenstiel, S. A1 - Kracht, M. A1 - Püschel, Gerhard Paul T1 - NF-kB-dependent IL-8 induction by prostaglandin EP2 receptors EP1 and EP4 JF - British journal of pharmacology : journal of The British Pharmacological Society N2 - Background and Purpose Recent studies suggested a role for PGE2 in the expression of the chemokine IL-8. PGE2 signals via four different GPCRs, EP1-EP4. The role of EP1 and EP4 receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP1 (HEK-EP1), EP4 (HEK-EP4) or both receptors (HEK-EP1 + EP4). Experimental Approach IL-8 mRNA and protein induction and IL-8 promoter and NF-?B activation were assessed in EP expressing HEK cells. Key Results In HEK-EP1 and HEK-EP1 + EP4 but not HEK or HEK-EP4 cells, PGE2 activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP1 + EP4 cells with an EP1-specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP4 agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP1 + EP4 cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE2. In HEK-EP1 + EP4 cells, PGE2-mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of I?B kinase. PGE2 activated NF-?B in HEK-EP1, HEK-EP4 and HEK-EP1 + EP4 cells. In HEK-EP1 + EP4 cells, simultaneous activation of both receptors was needed for maximal PGE2-induced NF-?B activation. PGE2-stimulated NF-?B activation by EP1 was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP4 was only blunted by Src-kinase inhibition. Conclusions and Implications These findings suggest that PGE2-mediated NF-?B activation by simultaneous stimulation of EP1 and EP4 receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction. KW - prostaglandin receptor KW - NF-?B KW - IL-8 transcription KW - signal transduction Y1 - 2013 U6 - https://doi.org/10.1111/j.1476-5381.2012.02182.x SN - 0007-1188 VL - 168 IS - 3 SP - 704 EP - 717 PB - Wiley-Blackwell CY - Hoboken ER - TY - GEN A1 - Camargo, Rodolfo Gonzalez A1 - Riccardi, Daniela Mendes dos Reis A1 - Ribeiro, Henrique Quintas Teixeira A1 - Carnevali Junior, Luiz Carlos A1 - Matos-Neto, Emidio Marques de A1 - Enjiu, Lucas A1 - Neves, Rodrigo Xavier A1 - Lima, Joanna Darck Carola Correia A1 - Figuerêdo, Raquel Galvão A1 - Alcântara, Paulo Sérgio Martins de A1 - Maximiano, Linda A1 - Otoch, José A1 - Batista Jr., Miguel Luiz A1 - Püschel, Gerhard Paul A1 - Seelaender, Marilia T1 - NF-kappa Bp65 and expression of its pro-inflammatory target genes are upregulated in the subcutaneous adipose tissue of cachectic cancer patients N2 - Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-κB). We have examined the gene expression of the subunits NF-κBp65 and NF-κBp50, as well as NF-κBp65 and NF-κBp50 binding, the gene expression of pro-inflammatory mediators under NF-κB control (IL-1β, IL-6, INF-γ, TNF-α, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-κBp65 and its target genes expression (TNF-α, IL-1β, MCP-1 and IκB-α) were significantly higher in cachectic cancer patients. Moreover, NF-κBp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-κB pathway plays a role in the promotion of WAT inflammation during cachexia. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 349 KW - cancer cachexia KW - inflammation KW - white adipose tissue KW - NF-κB KW - IκB Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-400163 ER - TY - JOUR A1 - Camargo, Rodolfo Gonzalez A1 - dos Reis Riccardi, Daniela Mendes A1 - Teixeira Ribeiro, Henrique Quintas A1 - Carnevali Junior, Luiz Carlos A1 - de Matos-Neto, Emidio Marques A1 - Enjiu, Lucas A1 - Neves, Rodrigo Xavier A1 - Carola Correia Lima, Joanna Darck A1 - Figueredo, Raquel Galvao A1 - Martins de Alcantara, Paulo Sergio A1 - Maximiano, Linda A1 - Otoch, Jose A1 - Batista Jr., Miguel Luiz A1 - Püschel, Gerhard Paul A1 - Seelaender, Marilia T1 - NF-kappa Bp65 and Expression of Its Pro-Inflammatory Target Genes Are Upregulated in the Subcutaneous Adipose Tissue of Cachectic Cancer Patients JF - Nutrients N2 - Cancer cachexia, of which the most notable symptom is severe and rapid weight loss, is present in the majority of patients with advanced cancer. Inflammatory mediators play an important role in the development of cachexia, envisaged as a chronic inflammatory syndrome. The white adipose tissue (WAT) is one of the first compartments affected in cancer cachexia and suffers a high rate of lipolysis. It secretes several cytokines capable of directly regulating intermediate metabolism. A common pathway in the regulation of the expression of pro-inflammatory cytokines in WAT is the activation of the nuclear transcription factor kappa-B (NF-B). We have examined the gene expression of the subunits NF-Bp65 and NF-Bp50, as well as NF-Bp65 and NF-Bp50 binding, the gene expression of pro-inflammatory mediators under NF-B control (IL-1, IL-6, INF-, TNF-, MCP-1), and its inhibitory protein, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IB-). The observational study involved 35 patients (control group, n = 12 and cancer group, n = 23, further divided into cachectic and non-cachectic). NF-Bp65 and its target genes expression (TNF-, IL-1, MCP-1 and IB-) were significantly higher in cachectic cancer patients. Moreover, NF-Bp65 gene expression correlated positively with the expression of its target genes. The results strongly suggest that the NF-B pathway plays a role in the promotion of WAT inflammation during cachexia. KW - cancer cachexia KW - inflammation KW - white adipose tissue KW - NF-B KW - IB Y1 - 2015 U6 - https://doi.org/10.3390/nu7064465 SN - 2072-6643 VL - 7 IS - 6 SP - 4465 EP - 4479 PB - MDPI CY - Basel ER - TY - GEN A1 - Gardemann, Andreas A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt T1 - Nervous control of liver metabolism and hemodynamics N2 - Content: Anatomy of hepatic innervation In vivo studies on the role of hepatic nerves Effects of hepatic nerves in isolated perfused liver Mechanism of action of sympathetic hepatic nerves T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 164 Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-51346 ER - TY - GEN A1 - Neuschäfer-Rube, Frank A1 - DeVries, Christa A1 - Hänecke, Kristina A1 - Jungermann, Kurt A1 - Püschel, Gerhard Paul T1 - Molecular cloning and expression of a prostaglandin E₂ receptor of the EP₃ϐ subtype from rat hepatocytes N2 - Rat hepatocytes have previously been reported to possess prostaglandin E₂ receptors of the EP₃-type (EP₃-receptors) that inhibit glucagonstimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP₃ϐ receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95% homology to the EP₃ϐ receptor from mouse mastocytoma. The amino- and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE₂ with an apparent Kd of 15 nM. PGE₂ > PGF₂α > PGD₂ competed for [³H]PGE₂ binding sites as did the EP₃ receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP₁ receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE₂ > misoprostol > PGF₂α inhibited the forskolin-elicited cAMP formation. Thus, the characteristics of the EP₃ϐ receptor of rat hepatocytes closely resemble those of the EP₃ϐ receptor of mouse mastocytoma. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 108 KW - Prostaglandin receptor KW - Hepatocyte (rat) KW - Molecular cloning and expression Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45830 ER - TY - JOUR A1 - Hocher, Berthold A1 - Haumann, Hannah A1 - Rahnenführer, Jan A1 - Reichetzeder, Christoph A1 - Kalk, Philipp A1 - Pfab, Thiemo A1 - Tsuprykov, Oleg A1 - Winter, Stefan A1 - Hofmann, Ute A1 - Li, Jian A1 - Püschel, Gerhard Paul A1 - Lang, Florian A1 - Schuppan, Detlef A1 - Schwab, Matthias A1 - Schaeffeler, Elke T1 - Maternal eNOS deficiency determines a fatty liver phenotype of the offspring in a sex dependent manner JF - Epigenetics : the official journal of the DNA Methylation Society N2 - Maternal environmental factors can impact on the phenotype of the offspring via the induction of epigenetic adaptive mechanisms. The advanced fetal programming hypothesis proposes that maternal genetic variants may influence the offspring's phenotype indirectly via epigenetic modification, despite the absence of a primary genetic defect. To test this hypothesis, heterozygous female eNOS knockout mice and wild type mice were bred with male wild type mice. We then assessed the impact of maternal eNOS deficiency on the liver phenotype of wild type offspring. Birth weight of male wild type offspring born to female heterozygous eNOS knockout mice was reduced compared to offspring of wild type mice. Moreover, the offspring displayed a sex specific liver phenotype, with an increased liver weight, due to steatosis. This was accompanied by sex specific differences in expression and DNA methylation of distinct genes. Liver global DNA methylation was significantly enhanced in both male and female offspring. Also, hepatic parameters of carbohydrate metabolism were reduced in male and female offspring. In addition, male mice displayed reductions in various amino acids in the liver. Maternal genetic alterations, such as partial deletion of the eNOS gene, can affect liver metabolism of wild type offspring without transmission of the intrinsic defect. This occurs in a sex specific way, with more detrimental effects in females. This finding demonstrates that a maternal genetic defect can epigenetically alter the phenotype of the offspring, without inheritance of the defect itself. Importantly, these acquired epigenetic phenotypic changes can persist into adulthood. KW - Epigenetics KW - eNOS KW - Fetal programming KW - fatty liver KW - metabolism Y1 - 2016 U6 - https://doi.org/10.1080/15592294.2016.1184800 SN - 1559-2294 SN - 1559-2308 VL - 11 SP - 539 EP - 552 PB - Routledge, Taylor & Francis Group CY - Philadelphia ER - TY - GEN A1 - Schäfer, Marjänn Helena A1 - Kakularam, Kumar Reddy A1 - Reisch, Florian A1 - Rothe, Michael A1 - Stehling, Sabine A1 - Heydeck, Dagmar A1 - Püschel, Gerhard Paul A1 - Kuhn, Hartmut T1 - Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1295 KW - eicosanoids KW - lipid peroxidation KW - oxidative stress KW - polyenoic fatty acids KW - erythropoiesis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-576491 SN - 1866-8372 IS - 1295 ER - TY - JOUR A1 - Schäfer, Marjänn Helena A1 - Kakularam, Kumar Reddy A1 - Reisch, Florian A1 - Rothe, Michael A1 - Stehling, Sabine A1 - Heydeck, Dagmar A1 - Püschel, Gerhard Paul A1 - Kuhn, Hartmut T1 - Male Knock-in Mice Expressing an Arachidonic Acid Lipoxygenase 15B (Alox15B) with Humanized Reaction Specificity Are Prematurely Growth Arrested When Aging JF - Biomedicines N2 - 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. KW - eicosanoids KW - lipid peroxidation KW - oxidative stress KW - polyenoic fatty acids KW - erythropoiesis Y1 - 2022 U6 - https://doi.org/10.3390/biomedicines10061379 SN - 2227-9059 VL - 10 SP - 1 EP - 22 PB - MDPI CY - Basel, Schweiz ET - 6 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Klauder, Julia A1 - Henkel-Oberländer, Janin T1 - Macrophages, Low-Grade Inflammation, Insulin Resistance and Hyperinsulinemia: A Mutual Ambiguous Relationship in the Development of Metabolic Diseases T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Metabolic derangement with poor glycemic control accompanying overweight and obesity is associated with chronic low-grade inflammation and hyperinsulinemia. Macrophages, which present a very heterogeneous population of cells, play a key role in the maintenance of normal tissue homeostasis, but functional alterations in the resident macrophage pool as well as newly recruited monocyte-derived macrophages are important drivers in the development of low-grade inflammation. While metabolic dysfunction, insulin resistance and tissue damage may trigger or advance pro-inflammatory responses in macrophages, the inflammation itself contributes to the development of insulin resistance and the resulting hyperinsulinemia. Macrophages express insulin receptors whose downstream signaling networks share a number of knots with the signaling pathways of pattern recognition and cytokine receptors, which shape macrophage polarity. The shared knots allow insulin to enhance or attenuate both pro-inflammatory and anti-inflammatory macrophage responses. This supposedly physiological function may be impaired by hyperinsulinemia or insulin resistance in macrophages. This review discusses the mutual ambiguous relationship of low-grade inflammation, insulin resistance, hyperinsulinemia and the insulin-dependent modulation of macrophage activity with a focus on adipose tissue and liver. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1279 KW - NAFLD/MAFLD KW - type 2 diabetes KW - obesity KW - vicious cycle KW - TLR signaling KW - M1/M2 differentiation KW - Akt pathway Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-570106 SN - 1866-8372 IS - 1279 SP - 1 EP - 30 ER - TY - JOUR A1 - Püschel, Gerhard A1 - Klauder, Julia A1 - Henkel-Oberländer, Janin T1 - Macrophages, low-grade inflammation, insulin resistance and hyperinsulinemia BT - A mutual ambiguous relationship in the development of metabolic diseases JF - Journal of Clinical Medicine : open access journal N2 - Metabolic derangement with poor glycemic control accompanying overweight and obesity is associated with chronic low-grade inflammation and hyperinsulinemia. Macrophages, which present a very heterogeneous population of cells, play a key role in the maintenance of normal tissue homeostasis, but functional alterations in the resident macrophage pool as well as newly recruited monocyte-derived macrophages are important drivers in the development of low-grade inflammation. While metabolic dysfunction, insulin resistance and tissue damage may trigger or advance pro-inflammatory responses in macrophages, the inflammation itself contributes to the development of insulin resistance and the resulting hyperinsulinemia. Macrophages express insulin receptors whose downstream signaling networks share a number of knots with the signaling pathways of pattern recognition and cytokine receptors, which shape macrophage polarity. The shared knots allow insulin to enhance or attenuate both pro-inflammatory and anti-inflammatory macrophage responses. This supposedly physiological function may be impaired by hyperinsulinemia or insulin resistance in macrophages. This review discusses the mutual ambiguous relationship of low-grade inflammation, insulin resistance, hyperinsulinemia and the insulin-dependent modulation of macrophage activity with a focus on adipose tissue and liver. KW - NAFLD/MAFLD KW - type 2 diabetes KW - obesity KW - vicious cycle KW - TLR signaling KW - M1/M2 differentiation KW - Akt pathway Y1 - 2022 U6 - https://doi.org/10.3390/jcm11154358 SN - 2077-0383 VL - 11 IS - 15 SP - 1 EP - 30 PB - MDPI CY - Basel, Schweiz ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Mentlein, Rolf A1 - Heymann, Eberhard T1 - Isolation and characterization of Dipeptidyl Peptidase IV from human placenta N2 - Human placenta is surprisingly rich in post-proline dipeptidyl peptidase activity. Among various cell fractions, microsomes have the highest specific activity. A homogeneous enzyme preparation is obtained in a six-step purification procedure. The final preparation appears homogeneous upon dodecyl sulfate electrophoresis, but analytical isoelectric focussing reveals various active bands with isoelectric points in the range of pH 3 - 4. The enzyme is a glycoprotein containing about 30% carbohydrate. Treatment with neuraminidase lowers the isoelectric points but does not reduce the heterogeneity of the band pattern. The subunit molecular weight is 120000 as estimated by dodecyl sulfate electrophoresis, whereas Mr of the native enzyme is > 200000, as can be concluded from gel filtration experiments. The purified dipeptidyl peptidase cleaves various synthetic and natural peptides, including substance P, kentsin, casomorphin and a synthetic renin inhibitor. In general, the specificity of the placenta peptidase is similar to that of post-proline dipeptidyl peptidase from other sources. Phenylalanylprolyl-P-naphthylamide (Km = 0.02 mM, I/ = 92 Ujmg) is the best substrate among various synthetic peptide derivatives. Only peptides with a free N-terminal amino group and proline, hydroxyproline, or alanine in position 2 of the N-terminal sequence are cieaved. However, X-Pro-Pro- . . . structures, e. g. as in bradykinin, are not attacked. 1 mM bis-(6nitrophenyI)phosphate or 1 mM diisopropylfluorophosphate completely inactivate the peptidase within 30 min at 30°C (pH 8). The peptidase is also completely inhibited by 1 mM Zn²⁺ and by other heavy metals. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 114 Y1 - 1982 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45875 ER - TY - JOUR A1 - Fayyaz, Susann A1 - Henkel, Janin A1 - Japtok, Lukasz A1 - Krämer, Stephanie A1 - Damm, Georg A1 - Seehofer, Daniel A1 - Püschel, Gerhard Paul A1 - Kleuser, Burkhard T1 - Involvement of sphingosine 1-phosphate in palmitate-induced insulin resistance of hepatocytes via the S1P(2) receptor subtype JF - Diabetologia : journal of the European Association for the Study of Diabetes (EASD) N2 - 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. 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. 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 S1P(2) 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 S1P(2), was not able to inhibit insulin signalling. These data indicate that palmitate is metabolised by hepatocytes to S1P, which acts via stimulation of the S1P(2) receptor to impair insulin signalling. In particular, S1P(2) inhibition could be considered as a novel therapeutic target for the treatment of insulin resistance. KW - FTY720 KW - Insulin signalling KW - Palmitate KW - S1P receptors KW - Sphingolipids KW - Sphingosine 1-phosphate Y1 - 2014 U6 - https://doi.org/10.1007/s00125-013-3123-6 SN - 0012-186X SN - 1432-0428 VL - 57 IS - 2 SP - 373 EP - 382 PB - Springer CY - New York ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Jungermann, Kurt T1 - Integration of function in the hepatic acinus : intercellular communication in neural and humoral control of liver metabolism N2 - Content: Architecture of the liver acinus Functional zonation of the liver acinus Topological organization of metabollc regulation in the acinus Topological organization of defense and organ structure regulation in the acinus T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 163 Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-51279 ER - TY - JOUR A1 - Manowsky, Julia A1 - Camargo, Rodolfo Gonzalez A1 - Kipp, Anna Patricia A1 - Henkel, Janin A1 - Püschel, Gerhard Paul T1 - Insulin-induced cytokine production in macrophages causes insulin resistance in hepatocytes JF - American journal of physiology : Endocrinology and metabolism N2 - 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 beta-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-1 beta, IL-8, CCL2, and OSM. The insulin-elicited induction of IL-1 beta was independent of the presence of endotoxin and most likely mediated by an insulin-dependent activation of NF-kappa B. 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 IKK beta, 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. KW - metabolic syndrome KW - type 2 diabetes KW - inflammation KW - macrophage KW - insulin KW - cytokines Y1 - 2016 U6 - https://doi.org/10.1152/ajpendo.00427.2015 SN - 0193-1849 SN - 1522-1555 VL - 310 SP - E938 EP - E946 PB - American Chemical Society CY - Bethesda ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Miura, Hisayuki A1 - Neuschäfer-Rube, Frank A1 - Jungermann, Kurt T1 - Inhibition by the protein kinase C activator 4β-phorbol 12-myristate 13-acetate of the prostaglandin F₂α-mediated and noradrenaline-mediated but not glucagon-mediated activation of glycogenolysis in rat liver N2 - In perfused rat livers, infusion of prostaglandin F₂α (PGF₂α) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13-myristate 14-acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF₂α but did not alter the effects of glucagon. In isolated rat hepatocytes PGF₂α, noradrenaline and glucagon activated glycogen phosphorylase but only PGF₂α and noradrenaline increased intracellular inositol 1,4,5-1risphosphalc (InsP₃). The noradrenaline- or PGF₂α-elicited activation of glycogen phosphorylase and increase in InsP₃ were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon-mediated enzyme activation was not affected. In contra\t to PMA, the phorbol ester 4a-phorbol 13,14-didecanoate. which does not activate protein kinase C, did not attenuate the PGF₂α- and noradrenaline-elicited stimulation of glucose output, glycogen phosphorylase and InsP, formation. Stimulation of InsP₃ formation by AlF₄⁻, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA. Plasma membranes purified from isolated hepatocytes had both a high-capacity, low-affinity and a low-capacity, high-affinity binding site for PGF₂α. The Kd of the high-capacity, low-affinity binding site was close to the concentration of PGF₂α that increased glycogen phosphorylase activity halfmaximally. Binding to the high-capacity, low-affinity binding site was enhanced by guanosine 5'- 0-(3-thio)triphosphate (GTP[S]). This high-capacity, low-affinity site might thus represent the receptor. The Bmax and Kd of the high-capacity site, as well as the enhancement by GTP[S] of PGF₂α binding to this site, remained unaffected by PMA pretreatment. It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP₃-mediated signal pathway from PGF₂α via a PGF₂α receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 115 Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45889 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Christ, Bruno T1 - Inhibition by PGE₂ of glucagon-induced increase in phosphoenolpyruvate carboxykinase mRNA and acceleration of mRNA degradation in cultured rat hepatocytes N2 - In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E₂ has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE₂ to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE₂ of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE₂ accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE₂ is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE₂ may reduce the hepatic gluconeogenic capacity via a Gᵢ-linked signal chain. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 045 KW - Prostaglandin E₂ KW - Glucagon KW - Phosphoenolpyruvate carboxykinase KW - Inflammation KW - mRNA degradation Y1 - 1994 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45792 ER - TY - INPR A1 - Seelaender, Marilia A1 - Laviano, A. A1 - Busquets, S. A1 - Püschel, Gerhard Paul A1 - Margaria, T. A1 - Batista Jr., Miguel Luiz T1 - Inflammation in Cachexia T2 - Mediators of inflammation Y1 - 2015 U6 - https://doi.org/10.1155/2015/536954 SN - 0962-9351 SN - 1466-1861 PB - Hindawi Publishing Corp. CY - New York ER - TY - JOUR A1 - Henkel, Janin A1 - Coleman, Charles Dominic A1 - Schraplau, Anne A1 - Jöhrens, Korinna A1 - Weber, Daniela A1 - Castro, Jose Pedro A1 - Hugo, Martin A1 - Schulz, Tim Julius A1 - Krämer, Stephanie A1 - Schürmann, Annette A1 - Püschel, Gerhard Paul T1 - Induction of Steatohepatitis (NASH) with Insulin Resistance in Wild-type B6 Mice by a Western-type Diet Containing Soybean Oil and Cholesterol JF - Molecular medicine N2 - Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of the metabolic syndrome. Many currently used animal models of NAFLD/NASH lack clinical features of either NASH or metabolic syndrome such as hepatic inflammation and fibrosis (e.g., high-fat diets) or overweight and insulin resistance (e.g., methionine-choline-deficient diets), or they are based on monogenetic defects (e.g., ob/ob mice). In the current study, a Western-type diet containing soybean oil with high n-6-PUFA and 0.75% cholesterol (SOD + Cho) induced steatosis, inflammation and fibrosis accompanied by hepatic lipid peroxidation and oxidative stress in livers of C57BL/6-mice, which in addition showed increased weight gain and insulin resistance, thus displaying a phenotype closely resembling all clinical features of NASH in patients with metabolic syndrome. In striking contrast, a soybean oil-containing Western-type diet without cholesterol (SOD) induced only mild steatosis but not hepatic inflammation, fibrosis, weight gain or insulin resistance. Another high-fat diet, mainly consisting of lard and supplemented with fructose in drinking water (LAD + Fru), resulted in more prominent weight gain, insulin resistance and hepatic steatosis than SOD + Cho, but livers were devoid of inflammation and fibrosis. Although both LAD + Fru-and SOD + Cho-fed animals had high plasma cholesterol, liver cholesterol was elevated only in SOD + Cho animals. Cholesterol induced expression of chemotactic and inflammatory cytokines in cultured Kupffer cells and rendered hepatocytes more susceptible to apoptosis. In summary, dietary cholesterol in the SOD + Cho diet may trigger hepatic inflammation and fibrosis. SOD + Cho-fed animals may be a useful disease model displaying many clinical features of patients with the metabolic syndrome and NASH. KW - Nonalcoholic Steatohepatitis (NASH) KW - Typical Western Diet KW - Nonalcoholic Fatty Liver Disease (NAFLD) KW - Dietary Cholesterol KW - Kupffer Cells Y1 - 2017 U6 - https://doi.org/10.2119/molmed.2016.00203 SN - 1076-1551 SN - 1528-3658 VL - 23 SP - 70 EP - 82 PB - Feinstein Inst. for Medical Research CY - Manhasset ER - TY - JOUR A1 - Fennekohl, Alexandra A1 - Lucas, Maria A1 - Püschel, Gerhard Paul T1 - Induction by interleukin 6 of G(s)-coupled prostaglandin E(2) receptors in rat hepatocytes mediating a prostaglandin e(2)-dependent inhibition of the hepatocyte's acute phase response N2 - Prostanoids, that are released from nonparenchymal liver cells in response to proinflammatory stimuli, are involved in the regulation of hepatic functions during inflammation. They exert their effects on their target cells via heptahelical receptors in the plasma membrane. For the 5 prostanoids prostaglandin E(2) (PGE(2)), prostaglandin F(2alpha), prostaglandin D(2) (PGD(2)), prostacyclin, and thromboxane A(2) there exist 8 receptors that are coupled to different heterotrimeric G proteins. These receptors are expressed differentially in the 4 principal liver cell types, i.e., hepatocytes, Kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells. It was intriguing, that the messenger RNA (mRNA) of none of the G(s)-coupled prostanoid receptors (DP-R, EP2-R, EP4-R, and IP-R) that can attenuate the inflammatory reaction were present in hepatocytes. The current study shows that the expression of the G(s)-coupled prostanoid receptors EP2-R, EP4-R, and DP-R, but not the IP-R, was efficiently and rapidly up-regulated by treatment of hepatocytes in vitro or rats in vivo with the key acute phase cytokine interleukin 6 (IL-6). In IL-6-treated hepatocytes PGE(2) in turn attenuated the IL-6-induced alpha(2)-macroglobulin formation via a cyclic adenosine monophosphate (cAMP)- dependent signal chain. The data indicate that an IL-6-mediated induction of the previously not expressed EP2-R and EP4- R on hepatocytes might establish a prostanoid-mediated feedback inhibition loop for the attenuation of the acute phase response. Y1 - 2000 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Nath, Annegret A1 - Jungermann, Kurt T1 - Increase of urate formation by stimulation of sympathetic hepatic nerves, circulating noradrenaline and glucagon inthe perfused rat liver N2 - In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the α1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 038 KW - Urate KW - Allantoin KW - Hepatic nerve KW - Catecholamine KW - Glucagon Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16728 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Oppermann, Martin A1 - Muschol, Waldemar A1 - Götze, Otto A1 - Jungermann, Kurt T1 - Increase of glucose and lactate output and decrease of flow by human anaphylatoxin C3a but not C5a in perfused rat liver N2 - The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 039 KW - Hepatic glucose balance KW - Hepatic lactate balance KW - Hepatic hemodynamics KW - Complement system KW - Anaphylatoxin Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16733 ER - TY - GEN A1 - Püschel, Gerhard Paul A1 - Hespeling, Ursula A1 - Oppermann, Martin A1 - Dieter, Peter T1 - Increase in prostanoid formation in rat liver macrophages (Kupffer cells) by human anaphylatoxin C3a N2 - Human anaphylatoxin C3a increases glycogenolysis in perfused rat liver. This action is inhibited by prostanoid synthesis inhibitors and prostanoid antagonists. Because prostanoids but not anaphylatoxin C3a can increase glycogenolysis in hepatocytes, it has been proposed that prostanoid formation in nonparenchymal cells represents an important step in the C3a-dependent increase in hepatic glycogenolysis. This study shows that (a) human anaphylatoxin C3a (0.1 to 10 mug/ml) dose-dependently increased prostaglandin D2, thromboxane B, and prostaglandin F2alpha formation in rat liver macrophages (Kupffer cells); (b) the C3a-mediated increase in prostanoid formation was maximal after 2 min and showed tachyphylaxis; and (c) the C3a-elicited prostanoid formation could be inhibited specifically by preincubation of C3a with carboxypeptidase B to remove the essential C-terminal arginine or by preincubation of C3a with Fab fragments of a neutralizing monoclonal antibody. These data support the hypothesis that the C3a-dependent activation of hepatic glycogenolysis is mediated by way of a C3a-induced prostanoid production in Kupffer cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 037 KW - lactate output KW - glucose KW - complement KW - flow KW - prostaglandin-f2-alpha Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-16716 ER - TY - JOUR A1 - Schieferdecker, Henrike L. A1 - Pestel, Sabine A1 - Püschel, Gerhard Paul A1 - Götze, Otto T1 - Increase by anaphylatoxin C5a of glucose output in perfused rat liver via prostanoids derived from nonparenchymal cells : direct action of prostaglandins and indirect action of thromboxane A(2) on hepatocytes N2 - In the perfused rat liver the anaphylatoxin C5a enhanced glucose output, reduced flow, and elevated prostanoid overflow. Because hepatocytes (HCs) do not express C5a receptors, the metabolic C5a actions must be indirect, mediated by e.g. prostanoids from Kupffer cells (KCs) and hepatic stellate cells (HSCs), which possess C5a receptors. Surprisingly, the metabolic C5a effects were not only impaired by the prostanoid synthesis inhibitor, indomethacin, but also by the thromboxane A(2) (TXA(2)) receptor antagonist, daltroban, even though HCs do not express TXA(2) receptors. TXA(2) did not induce prostaglandin (PG) or an unknown factor release from KCs or sinusoidal endothelial cells (SECs), which express TXA(2) receptors, because (1) daltroban did neither influence the C5a-induced release of prostanoids from cultured KCs nor the C5a-dependent activation of glycogen phosphorylase in KC/HC cocultures and because (2) the TXA(2) analog, U46619, failed to stimulate prostanoid release from cultured KCs or SECs or to activate glycogen phosphorylase in KC/HC or SEC/HC cocultures. In the perfused liver, Ca(2+)-deprivation inhibited not only flow reduction but also glucose output elicited by C5a to similar extents as daltroban. Similarly, in the absence of extracellular Ca(2+), flow reduction and glucose output induced by U46619 were almost completely prevented, whereas glucose output induced by the directly acting PGF(2alpha) was only slightly lowered. Thus, in the perfused rat liver PGs released after C5a- stimulation from KCs and HSCs directly activated glycogen phosphorylase in HCs, and TXA(2) enhanced glucose output indirectly mainly by causing hypoxia as a result of flow reduction. Y1 - 1999 ER - TY - JOUR A1 - Neuschäfer-Rube, Frank A1 - Hermosilla, Ricardo A1 - Rehwald, Matthias A1 - Ronnstrand, Lars A1 - Schülein, Ralf A1 - Wernstedt, Christer A1 - Püschel, Gerhard Paul T1 - Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site N2 - hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C- terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist- induced phosphorylation, interaction with beta-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370-382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389-Ser-390-Thr-391-Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary. Y1 - 2004 UR - http://www.biochemj.org/bj/379/0573/bj3790573.htm ER - TY - JOUR A1 - Neuschäfer-Rube, Frank A1 - Engemaier, Eva A1 - Koch, Sina A1 - Böer, Ulrike A1 - Püschel, Gerhard Paul T1 - Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor N2 - Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand- binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF(2alpha) and PGD(2). Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF(2alpha)-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation. Y1 - 2003 UR - http://www.biochemj.org/bj/371/0443/bj3710443.htm ER -