@article{vonLoeffelholzLieskeNeuschaeferRubeetal.2017, author = {von Loeffelholz, Christian and Lieske, Stefanie and Neuschaefer-Rube, Frank and Willmes, Diana M. and Raschzok, Nathanael and Sauer, Igor M. and K{\"o}nig, J{\"o}rg and Fromm, Martin F. and Horn, Paul and Chatzigeorgiou, Antonios and Pathe-Neuschaefer-Rube, Andrea and Jordan, Jens and Pfeiffer, Andreas F. H. and Mingrone, Geltrude and Bornstein, Stefan R. and Stroehle, Peter and Harms, Christoph and Wunderlich, F. Thomas and Helfand, Stephen L. and Bernier, Michel and de Cabo, Rafael and Shulman, Gerald I. and Chavakis, Triantafyllos and P{\"u}schel, Gerhard Paul and Birkenfeld, Andreas L.}, title = {The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism}, series = {Hepatology}, volume = {66}, journal = {Hepatology}, number = {2}, publisher = {Wiley}, address = {Hoboken}, issn = {0270-9139}, doi = {10.1002/hep.29089}, pages = {616 -- 630}, year = {2017}, abstract = {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.}, language = {en} } @article{PatheNeuschaeferRubeNeuschaeferRubeGenzetal.2015, author = {Pathe-Neuschaefer-Rube, Andrea and Neuschaefer-Rube, Frank and Genz, Lara and P{\"u}schel, Gerhard Paul}, title = {Botulinum Neurotoxin Dose-Dependently Inhibits Release of Neurosecretory Vesicle-Targeted Luciferase from Neuronal Cells}, series = {Alternatives to animal experimentation : ALTEX ; a journal for new paths in biomedical science}, volume = {32}, journal = {Alternatives to animal experimentation : ALTEX ; a journal for new paths in biomedical science}, number = {4}, publisher = {Springer}, address = {Heidelberg}, issn = {1868-596X}, pages = {297 -- 306}, year = {2015}, abstract = {Botulinum toxin is a bacterial toxin that inhibits neurotransmitter release from neurons and thereby causes a flaccid paralysis. It is used as drug to treat a number of serious ailments and, more frequently, for aesthetic medical interventions. Botulinum toxin for pharmacological applications is isolated from bacterial cultures. Due to partial denaturation of the protein, the specific activity of these preparations shows large variations. Because of its extreme potential toxicity, pharmacological preparations must be carefully tested for their activity. For the current gold standard, the mouse lethality assay, several hundred thousand mice are killed per year. Alternative methods have been developed that suffer from one or more of the following deficits: In vitro enzyme assays test only the activity of the catalytic subunit of the toxin. Enzymatic and cell based immunological assays are specific for just one of the different serotypes. The current study takes a completely different approach that overcomes these limitations: Neuronal cell lines were stably transfected with plasmids coding for luciferases of different species, which were N-terminally tagged with leader sequences that redirect the luciferase into neuro-secretory vesicles. From these vesicles, luciferases were released upon depolarization of the cells. The depolarization-dependent release was efficiently inhibited by botulinum toxin in a concentration range (1 to 100 pM) that is used in pharmacological preparations. The new assay might thus be an alternative to the mouse lethality assay and the immunological assays already in use.}, language = {en} } @article{CamargodosReisRiccardiTeixeiraRibeiroetal.2015, author = {Camargo, Rodolfo Gonzalez and dos Reis Riccardi, Daniela Mendes and Teixeira Ribeiro, Henrique Quintas and Carnevali Junior, Luiz Carlos and de Matos-Neto, Emidio Marques and Enjiu, Lucas and Neves, Rodrigo Xavier and Carola Correia Lima, Joanna Darck and Figueredo, Raquel Galvao and Martins de Alcantara, Paulo Sergio and Maximiano, Linda and Otoch, Jose and Batista Jr., Miguel Luiz and P{\"u}schel, Gerhard Paul and Seelaender, Marilia}, title = {NF-kappa Bp65 and Expression of Its Pro-Inflammatory Target Genes Are Upregulated in the Subcutaneous Adipose Tissue of Cachectic Cancer Patients}, series = {Nutrients}, volume = {7}, journal = {Nutrients}, number = {6}, publisher = {MDPI}, address = {Basel}, issn = {2072-6643}, doi = {10.3390/nu7064465}, pages = {4465 -- 4479}, year = {2015}, abstract = {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.}, language = {en} } @article{NeuschaeferRubeSchraplauScheweetal.2015, author = {Neuschaefer-Rube, Frank and Schraplau, Anne and Schewe, Bettina and Lieske, Stefanie and Kruetzfeldt, Julia-Mignon and Ringel, Sebastian and Henkela, Janin and Birkenfeld, Andreas L. and P{\"u}schel, Gerhard Paul}, title = {Arylhydrocarbon receptor-dependent mIndy (SIc13a5) induction as possible contributor to benzo[a]pyrene-induced lipid accumulation in hepatocytes}, series = {Toxicology}, volume = {337}, journal = {Toxicology}, publisher = {Elsevier}, address = {Clare}, issn = {0300-483X}, doi = {10.1016/j.tox.2015.08.007}, pages = {1 -- 9}, year = {2015}, abstract = {Non-alcoholic fatty liver disease is a growing problem in industrialized and developing countries. Hepatic lipid accumulation is the result of an imbalance between fatty acid uptake, fatty acid de novo synthesis, beta-oxidation and secretion of triglyceride-rich lipoproteins from the hepatocyte. A central regulator of hepatic lipid metabolism is cytosolic citrate that can either be derived from the mitochondrium or be taken up from the blood via the plasma membrane sodium citrate transporter NaCT, the product of the mammalian INDY gene (SLC13A5). mINDY ablation protects against diet-induced steatosis whereas mINDY expression is increased in patients with hepatic steatosis. Diet-induced hepatic steatosis is also enhanced by activation of the arylhyrocarbon receptor (AhR) both in humans and animal models. Therefore, the hypothesis was tested whether the mINDY gene might be a target of the AhR. In accordance with such a hypothesis, the AhR activator benzo[a]pyrene induced the mINDY expression in primary cultures of rat hepatocytes in an AhR-dependent manner. This induction resulted in an increased citrate uptake and citrate incorporation into lipids which probably was further enhanced by the benzo[a]pyrene-dependent induction of key enzymes of fatty acid synthesis. A potential AhR binding site was identified in the mINDY promoter that appears to be conserved in the human promoter. Elimination or mutation of this site largely abolished the activation of the mINDY promoter by benzo[a]pyrene. This study thus identified the mINDY as an AhR target gene. AhR-dependent induction of the mINDY gene might contribute to the development of hepatic steatosis. (C) 2015 Elsevier Ireland Ltd. All rights reserved.}, language = {en} } @article{SprussHenkelKanurietal.2012, author = {Spruss, Astrid and Henkel, Janin and Kanuri, Giridhar and Blank, Daniela and P{\"u}schel, Gerhard Paul and Bischoff, Stephan C. and Bergheim, Ina}, title = {Female mice are more susceptible to nonalcoholic fatty liver disease sex-specific regulation of the hepatic AMP-Activated protein Kinase-Plasminogen activator inhibitor 1 cascade, but not the hepatic endotoxin response}, series = {Molecular medicine}, volume = {18}, journal = {Molecular medicine}, number = {9}, publisher = {Feinstein Inst. for Medical Research}, address = {Manhasset}, issn = {1076-1551}, doi = {10.2119/molmed.2012.00223}, pages = {1346 -- 1355}, year = {2012}, abstract = {As significant differences between sexes were found in the susceptibility to alcoholic liver disease in human and animal models, it was the aim of the present study to investigate whether female mice also are more susceptible to the development of nonalcoholic fatty liver disease (NAFLD). Male and female C57BL/6J mice were fed either water or 30\% fructose solution ad libitum for 16 wks. Liver damage was evaluated by histological scoring. Portal endotoxin levels and markers of Kupffer cell activation and insulin resistance, plasminogen activator inhibitor 1 (PAI-1) and phosphorylated adenosine monophosphate-activated protein kinase (pAMPK) were measured in the liver. Adiponectin mRNA expression was determined in adipose tissue. Hepatic steatosis was almost similar between male and female mice; however, inflammation was markedly more pronounced in livers of female mice. Portal endotoxin levels, hepatic levels of myeloid differentiation primary response gene (88) (MyD88) protein and of 4-hydroxynonenal protein adducts were elevated in animals with NAFLD regardless of sex. Expression of insulin receptor substrate 1 and 2 was decreased to a similar extent in livers of male and female mice with NAFLD. The less pronounced susceptibility to liver damage in male mice was associated with a superinduction of hepatic pAMPK in these mice whereas, in livers of female mice with NAFLD, PAI-1 was markedly induced. Expression of adiponectin in visceral fat was significantly lower in female mice with NAFLD but unchanged in male mice compared with respective controls. In conclusion, our data suggest that the sex-specific differences in the susceptibility to NAFLD are associated with differences in the regulation of the adiponectin-AMPK-PAI-1 signaling cascade. Online address: http://www.molmed.Org doi: 10.2119/molmed.2012.00223}, language = {en} } @article{SchenkeSchjeidePuescheletal.2020, author = {Schenke, Maren and Schjeide, Brit-Maren and P{\"u}schel, Gerhard Paul and Seeger, Bettina}, title = {Analysis of motor neurons differentiated from human induced pluripotent stem cells for the use in cell-based Botulinum neurotoxin activity assays}, series = {Toxins}, volume = {12}, journal = {Toxins}, number = {5}, publisher = {MDPI}, address = {Basel}, issn = {2072-6651}, doi = {10.3390/toxins12050276}, pages = {20}, year = {2020}, abstract = {Botulinum neurotoxins (BoNTs) are potent neurotoxins produced by bacteria, which inhibit neurotransmitter release, specifically in their physiological target known as motor neurons (MNs). For the potency assessment of BoNTs produced for treatment in traditional and aesthetic medicine, the mouse lethality assay is still used by the majority of manufacturers, which is ethically questionable in terms of the 3Rs principle. In this study, MNs were differentiated from human induced pluripotent stem cells based on three published protocols. The resulting cell populations were analyzed for their MN yield and their suitability for the potency assessment of BoNTs. MNs produce specific gangliosides and synaptic proteins, which are bound by BoNTs in order to be taken up by receptor-mediated endocytosis, which is followed by cleavage of specific soluble N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins required for neurotransmitter release. The presence of receptors and substrates for all BoNT serotypes was demonstrated in MNs generated in vitro. In particular, the MN differentiation protocol based on Du et al. yielded high numbers of MNs in a short amount of time with high expression of BoNT receptors and targets. The resulting cells are more sensitive to BoNT/A1 than the commonly used neuroblastoma cell line SiMa. MNs are, therefore, an ideal tool for being combined with already established detection methods.}, language = {en} } @article{HenkelColemanSchraplauetal.2017, author = {Henkel, Janin and Coleman, Charles Dominic and Schraplau, Anne and J{\"o}hrens, Korinna and Weber, Daniela and Castro, Jose Pedro and Hugo, Martin and Schulz, Tim Julius and Kr{\"a}mer, Stephanie and Sch{\"u}rmann, Annette and P{\"u}schel, Gerhard Paul}, title = {Induction of Steatohepatitis (NASH) with Insulin Resistance in Wild-type B6 Mice by a Western-type Diet Containing Soybean Oil and Cholesterol}, series = {Molecular medicine}, volume = {23}, journal = {Molecular medicine}, publisher = {Feinstein Inst. for Medical Research}, address = {Manhasset}, issn = {1076-1551}, doi = {10.2119/molmed.2016.00203}, pages = {70 -- 82}, year = {2017}, abstract = {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.}, language = {en} }