@misc{HespelingPueschelJungermannetal.1995, author = {Hespeling, Ursula and P{\"u}schel, Gerhard Paul and Jungermann, Kurt and G{\"o}tze, Otto and Zwirner, J{\"o}rg}, title = {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}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-45909}, year = {1995}, abstract = {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.}, language = {en} } @misc{NeuschaeferRubeDeVriesHaeneckeetal.1994, author = {Neusch{\"a}fer-Rube, Frank and DeVries, Christa and H{\"a}necke, Kristina and Jungermann, Kurt and P{\"u}schel, Gerhard Paul}, title = {Molecular cloning and expression of a prostaglandin E₂ receptor of the EP₃ϐ subtype from rat hepatocytes}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-45830}, year = {1994}, abstract = {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.}, language = {en} } @misc{HenkelBuchheimDieckowCastroetal.2019, author = {Henkel, Janin and Buchheim-Dieckow, Katja and Castro, Jos{\´e} Pedro and Laeger, Thomas and Wardelmann, Kristina and Kleinridders, Andr{\´e} and J{\"o}hrens, Korinna and P{\"u}schel, Gerhard Paul}, title = {Reduced Oxidative Stress and Enhanced FGF21 Formation in Livers of Endurance-Exercised Rats with Diet-Induced NASH}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {807}, issn = {1866-8372}, doi = {10.25932/publishup-44238}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-442384}, pages = {17}, year = {2019}, abstract = {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.}, language = {en} } @article{HenkelBuchheimDieckowCastroetal.2019, author = {Henkel, Janin and Buchheim-Dieckow, Katja and Castro, Jos{\´e} Pedro and Laeger, Thomas and Wardelmann, Kristina and Kleinridders, Andr{\´e} and J{\"o}hrens, Korinna and P{\"u}schel, Gerhard Paul}, title = {Reduced Oxidative Stress and Enhanced FGF21 Formation in Livers of Endurance-Exercised Rats with Diet-Induced NASH}, series = {Nutrients}, volume = {11}, journal = {Nutrients}, number = {11}, publisher = {MDPI}, address = {Basel}, issn = {2072-6643}, doi = {10.3390/nu11112709}, pages = {15}, year = {2019}, abstract = {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.}, language = {en} } @article{GehreFlechnerKammereretal.2020, author = {Gehre, Christian and Flechner, Marie and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Coleman, Charles Dominic and P{\"u}schel, Gerhard Paul and Uhlig, Katja and Duschl, Claus}, title = {Real time monitoring of oxygen uptake of hepatocytes in a microreactor using optical microsensors}, series = {Scientific reports}, volume = {10}, journal = {Scientific reports}, number = {1}, publisher = {Macmillan Publishers Limited, part of Springer Nature}, address = {[London]}, issn = {2045-2322}, doi = {10.1038/s41598-020-70785-6}, pages = {12}, year = {2020}, abstract = {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.}, language = {en} } @misc{UhligGehreKammereretal.2018, author = {Uhlig, Katja and Gehre, Christian P. and Kammerer, Sarah and K{\"u}pper, Jan-Heiner and Coleman, Charles Dominic and P{\"u}schel, Gerhard Paul and Duschl, Claus}, title = {Real-time monitoring of oxygen consumption of hepatocytes in a microbioreactor}, series = {Toxicology letters}, volume = {295}, journal = {Toxicology letters}, publisher = {Elsevier}, address = {Clare}, issn = {0378-4274}, doi = {10.1016/j.toxlet.2018.06.652}, pages = {S115 -- S115}, year = {2018}, language = {en} } @misc{NeuschaeferRubePatheNeuschaeferRubePueschel2022, author = {Neusch{\"a}fer-Rube, Frank and Pathe-Neusch{\"a}fer-Rube, Andrea and P{\"u}schel, Gerhard Paul}, title = {Discrimination of the Activity of Low-Affinity Wild-Type and High-Affinity Mutant Recombinant BoNT/B by a SIMA Cell-Based Reporter Release Assay}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, publisher = {Universit{\"a}tsverlag Potsdam}, address = {Potsdam}, issn = {1866-8372}, doi = {10.25932/publishup-55803}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-558032}, pages = {1 -- 11}, year = {2022}, abstract = {Botulinum neurotoxin (BoNT) is used for the treatment of a number of ailments. The activity of the toxin that is isolated from bacterial cultures is frequently tested in the mouse lethality assay. Apart from the ethical concerns inherent to this assay, species-specific differences in the affinity for different BoNT serotypes give rise to activity results that differ from the activity in humans. Thus, BoNT/B is more active in mice than in humans. The current study shows that the stimulus-dependent release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-Gluc) was inhibited by clostridial and recombinant BoNT/A to the same extent, whereas both clostridial and recombinant BoNT/B inhibited the release to a lesser extent and only at much higher concentrations, reflecting the low activity of BoNT/B in humans. By contrast, the genetically modified BoNT/B-MY, which has increased affinity for human synaptotagmin, and the BoNT/B protein receptor inhibited luciferase release effectively and with an EC50 comparable to recombinant BoNT/A. This was due to an enhanced uptake into the reporter cells of BoNT/B-MY in comparison to the recombinant wild-type toxin. Thus, the SIMA-hPOMC1-26-Gluc cell assay is a versatile tool to determine the activity of different BoNT serotypes providing human-relevant dose-response data.}, language = {en} } @misc{MichaudSchjeideSchenkeSeegeretal.2022, author = {Michaud Schjeide, Brit-Maren and Schenke, Maren and Seeger, Bettina and P{\"u}schel, Gerhard Paul}, title = {Validation of a Novel Double Control Quantitative Copy Number PCR Method to Quantify Off-Target Transgene Integration after CRISPR-Induced DNA Modification}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, publisher = {Universit{\"a}tsverlag Potsdam}, address = {Potsdam}, issn = {1866-8372}, doi = {10.25932/publishup-56175}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-561755}, pages = {1 -- 14}, year = {2022}, abstract = {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.}, language = {en} } @article{PueschelKlauderHenkelOberlaender2022, author = {P{\"u}schel, Gerhard and Klauder, Julia and Henkel-Oberl{\"a}nder, Janin}, title = {Macrophages, low-grade inflammation, insulin resistance and hyperinsulinemia}, series = {Journal of Clinical Medicine : open access journal}, volume = {11}, journal = {Journal of Clinical Medicine : open access journal}, number = {15}, publisher = {MDPI}, address = {Basel, Schweiz}, issn = {2077-0383}, doi = {10.3390/jcm11154358}, pages = {1 -- 30}, year = {2022}, abstract = {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.}, language = {en} } @misc{PueschelKlauderHenkelOberlaender, author = {P{\"u}schel, Gerhard Paul and Klauder, Julia and Henkel-Oberl{\"a}nder, Janin}, title = {Macrophages, Low-Grade Inflammation, Insulin Resistance and Hyperinsulinemia: A Mutual Ambiguous Relationship in the Development of Metabolic Diseases}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1279}, issn = {1866-8372}, doi = {10.25932/publishup-57010}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-570106}, pages = {1 -- 30}, abstract = {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.}, language = {en} }