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  - 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  - GEN
A1  - Schenke, Maren
A1  - Schjeide, Brit-Maren
A1  - Püschel, Gerhard Paul
A1  - Seeger, Bettina
T1  - Analysis of motor neurons differentiated from human induced pluripotent stem cells for the use in cell-based Botulinum neurotoxin activity assays
T2  - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2  - 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.
T3  - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1083 
KW  - Botulinum neurotoxin
KW  - motor neurons
KW  - cell-based in vitro assay
KW  - potency assessment
KW  - induced pluripotent stem cells
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-472071
SN  - 1866-8372
IS  - 1083
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  - GEN
A1  - Pathe-Neuschäfer-Rube, Andrea
A1  - Neuschäfer-Rube, Frank
A1  - Haas, Gerald
A1  - Langoth-Fehringer, Nina
A1  - Püschel, Gerhard Paul
T1  - Cell-Based Reporter Release Assay to Determine the Potency of Proteolytic Bacterial Neurotoxins
T2  - Toxins
N2  - Despite the implementation of cell-based replacement methods, the mouse lethality assay is still frequently used to determine the activity of botulinum toxin (BoNT) for medical use. One explanation is that due to the use of neoepitope-specific antibodies to detect the cleaved BoNT substrate, the currently devised assays can detect only one specific serotype of the toxin. Recently, we developed a cell-based functional assay, in which BoNT activity is determined by inhibiting the release of a reporter enzyme that is liberated concomitantly with the neurotransmitter from neurosecretory vesicles. In theory, this assay should be suitable to detect the activity of any BoNT serotype. Consistent with this assumption, 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 cells) was inhibited by BoNT-A and-C. Furthermore, this was also inhibited by BoNT-B and tetanus toxin to a lesser extent and at higher concentrations. In order to provide support for the suitability of this technique in practical applications, a dose–response curve obtained with a pharmaceutical preparation of BoNT-A closely mirrored the activity determined in the mouse lethality assay. In summary, the newly established cell-based assay may represent a versatile and specific alternative to the mouse lethality assay and other currently established cell-based assays.
T3  - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 473 
KW  - botulinum toxin
KW  - BoNT
KW  - tetanus toxin
KW  - RRR
KW  - replacement
Y1  - 2018
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-418141
ER  - 
TY  - JOUR
A1  - Henkel, Janin
A1  - Neuschaefer-Rube, Frank
A1  - Pathe-Neuschaefer-Rube, Andrea
A1  - Püschel, Gerhard Paul
T1  - Aggravation by prostaglandin e-2 of interleukin-6-dependent insulin resistance in hepatocytes
N2  - Hepatic insulin resistance is a major contributor to fasting hyperglycemia in patients with metabolic syndrome and type 2 diabetes. Circumstantial evidence suggests that cyclooxygenase products in addition to cytokines might contribute to insulin resistance. However, direct evidence for a role of prostaglandins in the development of hepatic insulin resistance is lacking. Therefore, the impact of prostaglandin E-2 (PGE(2)) alone and in combination with interleukin-6 (IL-6) on insulin signaling was studied in primary hepatocyte cultures. Rat hepatocytes were incubated with IL-6 and/or PGE(2) and subsequently with insulin. Glycogen synthesis was monitored by radiochemical analysis; the activation state of proteins of the insulin receptor signal chain was analyzed by western blot with phosphospecific antibodies. In hepatocytes, insulin-stimulated glycogen synthesis and insulin-dependent phosphorylation of Akt-kinase were attenuated synergistically by prior incubation with IL-6 and/or PGE(2) while insulin receptor autophosphorylation was barely affected. IL-6 but not PGE(2) induced suppressors of cytokine signaling (SOCS3). PGE(2) but not IL-6 activated extracellular signal-regulated kinase 1/2 (ERK1/2) persistently. Inhibition of ERK1/2 activation by PD98059 abolished the PGE(2)-dependent but not the IL-6-dependent attenuation of insulin signaling. In HepG2 cells expressing a recombinant EP3-receptor, PGE(2) pre-incubation activated ERK1/2, caused a serine phosphorylation of insulin receptor substrate 1 (IRS1), and reduced the insulin-dependent Akt-phosphorylation. Conclusion: PGE(2) might contribute to hepatic insulin resistance via an EP3-receptor-dependent ERK1/2 activation resulting in a serine phosphorylation of insulin receptor substrate, thereby preventing an insulin-dependent activation of Akt and glycogen synthesis. Since different molecular mechanisms appear to be employed, PGE(2) may synergize with IL-6, which interrupted the insulin receptor signal chain, principally by an induction of SOCS, namely SOCS3.
Y1  - 2009
UR  - http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291527-3350
U6  - https://doi.org/10.1002/Hep.23064
SN  - 0270-9139
ER  - 
TY  - JOUR
A1  - Neuschäfer-Rube, Frank
A1  - Oppermann, Martin
A1  - Möller, Ulrike
A1  - Böer, Ulrike
A1  - Püschel, Gerhard Paul
T1  - Agonist-induced phosphorylation by G protein-coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E(2) receptor hybrid
N2  - Prostaglandin E(2) receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G(q), EP2 and EP4 to G(s), and EP3 to G(i). The EP4-R, in contrast to the EP3beta-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G(i)-coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E(2), or by the EP3-R agonist M&B28767 was investigated in COS-7 cells expressing FLAG-epitope-tagged rat EP3beta-R (rEP3beta-R), hEP4-R, or rEP3hEP4- Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG- rEP3beta-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E(2) stimulated phosphorylation of the FLAG- hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&B28767 induced a time- and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAG-rEP3beta-R. Agonist-induced phosphorylation of the FLAG- hEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R- expressing COS-7 cells augmented the M&B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R.
Y1  - 1999
SN  - 1521-0111
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  - 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  - 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  -