Wolfgang Schechinger

Publications

• 6.55

  Impact points

  Erratum to: The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes.

  J Giebelstein, G Poschmann, K Højlund, W Schechinger, J W Dietrich, K Levin, H Beck-Nielsen, K Podwojski, K Stühler, H E Meyer, H H Klein

  Diabetologia. 04/2012;
• 6.55

  Impact points

  The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes.

  J Giebelstein, G Poschmann, K Højlund, W Schechinger, J W Dietrich, K Levin, H Beck-Nielsen, K Podwojski, K Stühler, H E Meyer, H H Klein

  Diabetologia. 04/2012; 55(4):1114-27.

  The molecular mechanisms underlying insulin resistance in skeletal muscle are incompletely understood. Here, we aimed to obtain a global picture of changes in protein abundance in skeletal muscle in obesity and type 2 diabetes, and those associated with whole-body measures of insulin action. Skeleta... [more] The molecular mechanisms underlying insulin resistance in skeletal muscle are incompletely understood. Here, we aimed to obtain a global picture of changes in protein abundance in skeletal muscle in obesity and type 2 diabetes, and those associated with whole-body measures of insulin action. Skeletal muscle biopsies were obtained from ten healthy lean (LE), 11 obese non-diabetic (OB), and ten obese type 2 diabetic participants before and after hyperinsulinaemic-euglycaemic clamps. Quantitative proteome analysis was performed by two-dimensional differential-gel electrophoresis and tandem-mass-spectrometry-based protein identification. Forty-four protein spots displayed significant (p < 0.05) changes in abundance by at least a factor of 1.5 between groups. Several proteins were identified in multiple spots, suggesting post-translational modifications. Multiple spots containing glycolytic and fast-muscle proteins showed increased abundance, whereas spots with mitochondrial and slow-muscle proteins were downregulated in the OB and obese type 2 diabetic groups compared with the LE group. No differences in basal levels of myosin heavy chains were observed. The abundance of multiple spots representing glycolytic and fast-muscle proteins correlated negatively with insulin action on glucose disposal, glucose oxidation and lipid oxidation, while several spots with proteins involved in oxidative metabolism and mitochondrial function correlated positively with these whole-body measures of insulin action. Our data suggest that increased glycolytic and decreased mitochondrial protein abundance together with a shift in muscle properties towards a fast-twitch pattern in the absence of marked changes in fibre-type distribution contribute to insulin resistance in obesity with and without type 2 diabetes. The roles of several differentially expressed or post-translationally modified proteins remain to be elucidated.
• 6.55

  Impact points

  In vitro phage display in a rat beta cell line: a simple approach for the generation of a single-chain antibody targeting a novel beta cell-specific epitope.

  S Ueberberg, D Ziegler, W Schechinger, J W Dietrich, S Akinturk, H H Klein, S Schneider

  Diabetologia. 04/2010; 53(7):1384-94.

  The aim of the present study was to evaluate in vitro phage display in a beta cell line as a novel strategy for the isolation of beta cell-specific agents/biomarkers. A single-chain antibody (SCA) library was pre-incubated with AR42J cells in order to eliminate SCAs with exocrine binding properties.... [more] The aim of the present study was to evaluate in vitro phage display in a beta cell line as a novel strategy for the isolation of beta cell-specific agents/biomarkers. A single-chain antibody (SCA) library was pre-incubated with AR42J cells in order to eliminate SCAs with exocrine binding properties. It was then panned against INS-1 cells to select beta cell-targeted antibodies. By these means, we isolated a novel antibody, SCA B5, that binds rapidly (6.0 min) and with a 450-fold higher specificity to beta cells relative to exocrine cells. We estimated for SCA B5 a binding affinity in the low micromol/l range and 858 binding sites per beta cell. Confocal microscopy showed binding to the beta cell surface and confirmed subsequent internalisation. Moreover, staining of rat and human pancreatic tissue sections with SCA B5 suggests that the target epitope is presented in pancreatic beta cells of different origins. Infrared imaging revealed that labelling of beta cells with tracer SCA B5 is strictly dependent on beta cell mass. With competition assays we excluded insulin, glutamate decarboxylase, C-peptide and islet amyloid polypeptide as SCA B5 targets. In accordance with these predictions, SCA B5 homed in vivo highly selectively to normal beta cells and dysfunctional beta cells of diabetic rats. Moreover, accumulation of radioactively labelled SCA B5 in the pancreas was reduced by 80% after pre-injection with unlabelled SCA B5, thereby confirming the specific uptake in the pancreas. We report a simple strategy for the generation of an SCA targeting a novel beta cell-specific epitope.

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• Human beta-glucuronidase mutants with elevated enzymatic activity under physiological conditions and method for identifying such

  Roffler Steve, Wu Chia-Hung, Schechinger Wolfgang, Chen Kai-Chuan, Prijovic Zeljko M

  Ref. No: US20100129367

  Year: 01/2010

  A number of human beta-glucuronidase variants having higher enzymatic activity at physiological pH as compared with wild-type beta-glucuronidase and uses thereof in prodrug therapy. Also disclosed herein is a method for identifying enzyme variants having elevated enzymatic activity using a mammalian... [more] A number of human beta-glucuronidase variants having higher enzymatic activity at physiological pH as compared with wild-type beta-glucuronidase and uses thereof in prodrug therapy. Also disclosed herein is a method for identifying enzyme variants having elevated enzymatic activity using a mammalian surface display system.
• 8.51

  Impact points

  Generation of novel single-chain antibodies by phage-display technology to direct imaging agents highly selective to pancreatic {beta}- or {alpha}-cells in vivo.

  Sandra Ueberberg, Juris J Meier, Carmen Waengler, Wolfgang Schechinger, Johannes W Dietrich, Andrea Tannapfel, Inge Schmitz, Ralf Schirrmacher, Manfred Köller, Harald H Klein, Stephan Schneider

  Diabetes. 08/2009;

  Objective: Non-invasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet-cells in vivo. Research Design and Methods: In ord... [more] Objective: Non-invasive determination of pancreatic beta-cell mass in vivo has been hampered by the lack of suitable beta-cell specific imaging agents. This report outlines an approach for the development of novel ligands homing selectively to islet-cells in vivo. Research Design and Methods: In order to generate agents specifically binding to pancreatic islets, a phage-library was screened for single-chain-antibodies (SCAs) on rat islets using two different approaches: (1) The library was injected into rats in vivo, and islets were isolated after a circulation time of 5 minutes. (2) Pancreatic islets were directly isolated, and the library was panned in the islets in vitro. Subsequently, the identified SCAs were extensively characterized in vitro and in vivo. Results: We report the generation of SCAs that bind highly selective to either beta- or alpha-cells. These SCAs are internalised by target cells, disappear rapidly from the vasculature and exert no toxicity in vivo. Specific binding to beta- or alpha-cells was detected in cell-lines in vitro, in rats in vivo, and in human tissue in situ. Electron microscopy demonstrated binding of SCAs to the endoplasmatic reticulum and the secretory granules. Finally, in a biodistribution study the labeling intensity derived from [(125)I]-labeled SCAs after i.v. administration in rats strongly predicted the beta-cell mass and was inversely related to the glucose excursions during an intraperitoneal glucose tolerance test. Conclusions: Our data provide strong evidence that the presented SCAs are highly specific for pancreatic beta-cells and enable imaging and quantification in vivo.
• 6.52

  Impact points

  Directed Evolution of a Lysosomal Enzyme with Enhanced Activity at Neutral pH by Mammalian Cell-Surface Display.

  Kai-Chuan Chen, Chia-Hung Wu, Chuan-Yuan Chang, Wei-Cheng Lu, Qingzong Tseng, Zeljko M Prijovich, Wolfgang Schechinger, Yen-Chywan Liaw, Yu-Lin Leu, Steve R Roffler

  Chemistry & biology. 01/2009; 15(12):1277-86.

  Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity ba... [more] Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.
• 1.05

  Impact points

  Signalization of Akt/PKB in the placenta, skeletal muscle and adipose tissue of preeclampsia patients.

  Rafael Bueno Orcy, Sabrina Schroeder, Sérgio Hofmeister Martins-Costa, José Geraldo Lopes Ramos, Wolfgang Schechinger, Harald Klein, Ilma Simoni Brum, Helena von Eye Corleta, Edison Capp

  Gynecologic and obstetric investigation. 01/2008; 66(4):231-6.

  Preeclampsia (PE) is a significant cause of fetal and maternal mortality around the world and there is evidence that insulin resistance has been implicated in the pathophysiology of PE. The Akt/PKB pathway is stimulated by insulin and performs several vital functions relative to growth, survival and... [more] Preeclampsia (PE) is a significant cause of fetal and maternal mortality around the world and there is evidence that insulin resistance has been implicated in the pathophysiology of PE. The Akt/PKB pathway is stimulated by insulin and performs several vital functions relative to growth, survival and cellular metabolism. To investigate the basal expression of Akt/PKB, HSP90 expression, proteins that regulate Akt/PKB activity and substrate in the placenta, skeletal muscle and adipocytes of normal and PE parturient. Samples were collected from 17 normal patients and 17 PE patients, and analyzed by Western blot to quantify the protein expression involved in signaling cascade of Akt/PKB. Total Akt/PKB expression for normal placentas was 1.85 (1.07-3.12) and 1.53 (1.27-3.08) in PE (p = 1.00); in the adipose tissue of normal placentas it was 1.10 (0.53-1.73) and 1.66 (0.83-2.00) in PE (p = 0.37). There was no difference in the Akt/PKB pathway, in basal state, in placentas and skeletal muscle of normal and PE patients. However, defects in this signaling pathway as pathophysiology of PE cannot be excluded because it is necessary to analyze this pathway during stimulation.
• 2.16

  Impact points

  Synthesis and evaluation of a glibenclamide glucose-conjugate: a potential new lead compound for substituted glibenclamide derivatives as islet imaging agents.

  S Schneider, S Ueberberg, A Korobeynikov, W Schechinger, C Schwanstecher, M Schwanstecher, H H Klein, E Schirrmacher

  Regulatory peptides. 04/2007; 139(1-3):122-7.

  The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes i... [more] The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.
• 6.55

  Impact points

  Prolonged glucose infusion into conscious rats inhibits early steps in insulin signalling and induces translocation of GLUT4 and protein kinase C in skeletal muscle.

  B Houdali, V Nguyen, H P T Ammon, M Haap, W Schechinger, F Machicao, K Rett, H U Häring, E D Schleicher

  Diabetologia. 04/2002; 45(3):356-68.

  AIMS/HYPOTHESIS: Previous studies on diabetic patients have shown that hyperglycaemia increases glucose uptake in an apparently insulin-independent manner. However, the molecular mechanism has not been clarified. METHODS: We studied rats receiving continuous glucose infusion to address this question... [more] AIMS/HYPOTHESIS: Previous studies on diabetic patients have shown that hyperglycaemia increases glucose uptake in an apparently insulin-independent manner. However, the molecular mechanism has not been clarified. METHODS: We studied rats receiving continuous glucose infusion to address this question. In this animal model, rats accommodate systemic glucose oversupply and rapidly develop insulin resistance. RESULTS: Glucose infusion increased both plasma glucose and insulin concentrations to peak after one day. In spite of continuous glucose infusion normoglycaemia was reached after 5 days while insulin concentrations remained higher. Focusing our studies in day 2 (hyperglycaemia/hyperinsulinaemia) and day 5 (normoglycaemia/hyperinsulinaemia) we found, particularly in day 5, that the early steps of the insulin signalling cascade in skeletal muscle of glucose-infused rats were not more activated when compared to control animals as assessed by a comparable phosphorylation of the insulin receptor, IRS-1 and PKB and by an unaltered IRS-1-associated Ptd(Ins) 3' kinase activity. Continuous glucose infusion induced GLUT4 protein expression and translocation to the plasma membrane while neither expression nor translocation of GLUT1 was affected. Translocation of PKC- betaI, - betaII (> threefold) and -alpha, -theta (to a lesser extent) to the plasma membrane was significantly induced after 2 days but not after 5 days of glucose infusion when normoglycaemia was reached. CONCLUSIONS/INTERPRETATION: Our data support the hypothesis that continuous glucose infusion induces translocation of GLUT4 while the early steps of the insulin signalling cascade were not increased. These effects could be mediated by activation of PKC.

• Synthesis and evaluation of a glibenclamide glucose-conjugate: A potential new lead compound for substituted glibenclamide derivatives as islet imaging agents

  S. Schneider, S. Ueberberg, A. Korobeynikov, W. Schechinger, C. Schwanstecher, M. Schwanstecher, H.H. Klein, E. Schirrmacher

  Regulatory Peptides.

  The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes i... [more] The search for novel SUR1-ligands originates from the idea to influence the in vivo behaviour by adding new structural moieties to the glibenclamide structure while preserving its binding affinity. Important application of novel conjugates might be their use as radioactively labelled tracer probes in the non-invasive investigation of the islet mass. It is known that the imaging quality of a tracer could be improved by increasing its hydrophilicity, which leads to a reduced plasma protein binding and diminished the unspecific uptake by various organs. In this study the glucose molecule was chosen as a substitute of glibenclamide to enhance hydrophilicity. As expected glucose conjugation leads to a 12-fold increase of the hydrophilicity. In vitro evaluation showed that the conjugate binds with high affinity to SUR1. Interestingly, in vivo the hypoglycaemic action of the conjugate was of significant shorter duration compared to glibenclamide. In accordance, the conjugate was cleared much faster from the blood stream, due to a significant lower plasma protein binding. In conclusion, glycosylation proved to be a powerful tool for the development of a high affinity glibenclamide ligand with completely different pharmacodynamics. Therefore, the glucose-conjugate could be a potential lead compound for the design of substituted glibenclamide derivatives as islet imaging ligands.