TY - JOUR A1 - Wiese, Stefanie A1 - Gaertner, Sonja A1 - Rawel, Harshadrai Manilal A1 - Winterhalter, Peter A1 - Kulling, Sabine E. T1 - Protein interactions with cyanidin-3-glucoside and its influence on alpha-amylase activity N2 - BACKGROUND: Recent studies indicate that the bioavailability of anthocyanins is extremely low. One of the possible reasons could be their binding to proteins. Therefore, the binding affinity of cyanidin-3-glucoside (Cy3glc) to HSA and alpha-amylase was investigated by the quenching of protein tryptophan fluorescence. From data obtained, the binding constants and the free Gibbs energy were calculated. The changes in conformation of the proteins tested were studied with circular dichroism and the influence of binding on alpha-amylase activity determined. RESULTS: Cy3glc quenched the tryptophan fluorescence and upon ligand binding a change in protein structure was observed related to the corresponding decrease in the et-amylase activity. The association constants of 25 to 77 x 10(3) L mol(-1) were calculated for different proteins, indicating weak interactions of non-covalent nature. Competitive binding with HSA in the presence of 8-anilino-1-naphthalene sulfonic acid suggest involvement of hydrophobic interactions, in the case of HSA the possible site being subdomain IIA. CONCLUSION: The strongest affinity of Cy3glc for HSA being at pH 7 underlines its potential in transport and distribution of the phenolic compounds in organisms. An influence on salivary amylase activity is possible when drinking berry juices with high anthocyanins content. Y1 - 2009 UR - http://www3.interscience.wiley.com/journal/1294/home U6 - https://doi.org/10.1002/Jsfa.3407 SN - 0022-5142 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 - Tschumi, Sibylle A1 - Nagl, Britta A1 - Simonetti, Giacomo Domenico A1 - von Vigier, R. A1 - Uehlinger, Dominik A1 - Lauterburg, Bernhard A1 - Utsch, Boris T1 - Symptomatic pseudotumor cerebri : evidence for toxic vitamin A levels in patients with chronic renal insufficiency? Y1 - 2009 UR - http://www.springerlink.com/content/100382 U6 - https://doi.org/10.1007/s00467-009-1132-y SN - 0931-041X ER - TY - JOUR A1 - Thierbach, René A1 - Drewes, Gunnar A1 - Fusser, Markus A1 - Wolfrum, Kathrin A1 - Epe, Bernd A1 - Ristow, Michael A1 - Steinberg, Pablo T1 - A role for iron-sulfur cluster proteins in DNA repair Y1 - 2009 UR - http://www.springerlink.com/content/100530 U6 - https://doi.org/10.1007/s00210-009-0404-1 SN - 0028-1298 ER - TY - JOUR A1 - Ruefer, Corinna E. A1 - Kulling, Sabine E. A1 - Moeseneder, Jutta A1 - Winterhalter, Peter A1 - Bub, Achim T1 - Role of plasma lipoproteins in the transport of the soyabean isoflavones daidzein and daidzein-7-O-beta-D- glucoside N2 - Isoflavone intake is associated with various properties beneficial to human health which are related to their antioxidant activity, for example, to their ability to increase LDL oxidation resistance. However, the distribution of isoflavones among plasma lipoproteins has not yet been elucidated in vivo. Therefore, the objective of the present study was to investigate the association between daidzein (DAI) and lipoproteins in human plasma upon administration of the aglycone and glucoside form. Five men aged 22-30 years participated in a randomised, double-blind study in cross-over design. After ingestion of DAI and daidzein-7-O-beta-D-glucoside (DG) (1 mg DAI aglycone equivalents/kg body weight) blood samples were drawn before isoflavone administration as well as 1, 2, 3, 4.5, 6, 8, 10, 12, 24 and 48 h post-dose. Concentrations of DAI in the different lipoprotein fractions (chylomicrons, VLDL, LDL, HDL) and in the non-lipoprotein fraction were analysed using isotope dilution capillary GUMS. The lipoprotein fraction profiles were similar for all subjects and resembled those obtained for plasma in our previously published study. The lipoprotein distribution based on the area under the concentration-time profiles from 0 h to infinity in the different fractions were irrespective of the administered form: non-lipoprotein fraction (53%) > LDL (20%) > HDL (14%) > VLDL (9-5%) > chylomicrons (2-5%). Of DAI present in plasma, 47% was associated to lipoproteins. Concentrations in the different lipoprotein fractions as well as in the non-lipoprotein fraction were always higher after the ingestion of DG than of DAI. Taken together, these results demonstrate an association between isoflavones and plasma lipoproteins in vivo. Y1 - 2009 UR - http://journals.cambridge.org/action/displayJournal?jid=BJN U6 - https://doi.org/10.1017/S0007114509297224 SN - 0007-1145 ER - TY - JOUR A1 - Nagl, Britta A1 - Loui, Andrea A1 - Raila, Jens A1 - Felderhoff-Mueser, Ursula A1 - Obladen, Michael A1 - Schweigert, Florian J. T1 - Urinary vitamin A excretion in very low birth weight infants N2 - Vitamin A (VA) deficiency in very low birth weight (VLBW) infants is associated with an increased risk for disorders related to kidney and lung maturation and function. VA losses through increased urinary retinol (ROH) excretion might contribute to this deficiency risk. The mechanism accounting for ROH loss in the urine has not yet been clarified. The aim of this study was to assess the excretion of ROH, retinol-binding protein 4 (RBP4) and transthyretin (TTR) in urine from VLBW infants in comparison with that in term infants in relation to kidney function. Urine specimens were collected from 15 VLBW infants (birth weight < 1,500 g) as well as from 20 term infants during the first 2 days after birth. ROH in urine was detectable in 14 of the 15 VLBW infants at a median concentration of 234 nmol/g creatinine. In the group of term infants, 17 of the 20 excreted ROH, but at an approximately five-times lower concentration (P<0.001). Excretion of RBP4 and TTR was also much higher in VLBW infants (both P<0.001). The urinary ROH excretion in VLBW infants may be related to the impaired tubular handling of its carrier proteins RBP4 and TTR. Thus, ROH excretion might contribute to an increased risk of VA deficiency, especially in VLBW infants. Y1 - 2009 UR - http://www.springerlink.com/content/100382 U6 - https://doi.org/10.1007/s00467-008-0965-0 SN - 0931-041X ER - TY - JOUR A1 - Mueller, Carsten A1 - Ullmann, Kristina A1 - Wilkens, Andrea A1 - Winterhalter, Peter A1 - Toyokuni, Shinya A1 - Steinberg, Pablo T1 - Potent antioxidative activity of vineatrol (R) 30 grapevine-shoot extract N2 - The health promoting effects of a grapevine-shoot extract named Vineatrol (R) 30, which contains resveratrol (Resv) as well as considerable amounts of Resv oligomers, have recently been investigated. In the present study, we analyzed the free radical scavenging capacity, the ability to inhibit lipid peroxidation, and the capacity to enhance the human glutathione peroxidase 1 (GPx) and the human superoxide dismutase 1 (SOD) gene promoter activities of Vineatrol (R) 30. Vineatrol (R) 30 was able to scavenge the 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation and led to concentration-dependent inhibition of lipid peroxidation, Vineatrol (R) 30 not being superior to Resv alone in both cases. Vineatrol (R) 30 also enhanced the gene promoter activities of human GPx and SOD expressed in V79 cells, whereas this effect could not be demonstrated for Resv. In summary, the results presented in this study show that the Vineatrol (R) 30 grapevine-shoot extract is a free radical scavenger and potent antioxidant at non- eytotoxic concentrations. Y1 - 2009 UR - http://www.jstage.jst.go.jp/browse/bbb U6 - https://doi.org/10.1271/Bbb.90213 SN - 0916-8451 ER - TY - JOUR A1 - Mortensen, Alicja A1 - Kulling, Sabine E. A1 - Schwartz, Heidi A1 - Rowland, Ian A1 - Ruefer, Corinna E. A1 - Rimbach, Gerald A1 - Cassidy, Aedin A1 - Magee, Pamela A1 - Millar, Julie A1 - Hall, Wendy L. A1 - Kramer Birkved, Franziska A1 - Sorensen, Ilona K. A1 - Sontag, Gerhard T1 - Analytical and compositional aspects of isoflavones in food and their biological effects N2 - This paper provides an overview of analytical techniques used to determine isoflavones (IFs) in foods and biological fluids with main emphasis on sample preparation methods. Factors influencing the content of IFs in food including processing and natural variability are summarized and an insight into IF databases is given. Comparisons of dietary intake of IFs in Asian and Western populations, in special subgroups like vegetarians, vegans, and infants are made and our knowledge on their absorption, distribution, metabolism, and excretion by the human body is presented. The influences of the gut microflora, age, gender, background diet, food matrix, and the chemical nature of the IFs on the metabolism of IFs are described. Potential mechanisms by which IFs may exert their actions are reviewed, and genetic polymorphism as determinants of biological response to soy IFs is discussed. The effects of IFs on a range of health outcomes including atherosclerosis, breast, intestinal, and prostate cancers, menopausal symptoms, bone health, and cognition are reviewed on the basis of the available in vitro, in vivo animal and human data. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/jhome/109582333 U6 - https://doi.org/10.1002/mnfr.200800478 SN - 1613-4125 ER - TY - JOUR A1 - Maeder, Jens A1 - Rawel, Harshadrai Manilal A1 - Kroh, Lothar W. T1 - Composition of phenolic compounds and glycoalkaloids alpha-solanine and alpha-chaconine during commercial potato processing N2 - The influence of a commercial production process for dehydrated potato flakes on the content of free phenolic compounds, total phenolics, and glycoalkaloids in potatoes during the subsequent processing steps was determined. Processing byproducts, such as potato peel (steam peeling), mashed potato residues, and side streams (blanching and cooking waters), have also been investigated. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify caffeic acid, gallic acid, ferulic acid, p-coumaric acid, p-hydoxybenzoic acid, protocatechuic acid, vanillic acid, catechin, and three isomers of caffeoylquinic acid: chlorogenic, neochlorogenic and cryptochlorogenic acid. Determination of the glycoalkaloids a-solanine and alpha-chaconine was performed by using a high- performance thin-layer chromatography (HPTLC) method. The deliverables reveal that processing potatoes to potato flakes remarkably diminishes the content of the analyzed compounds, mainly due to peeling and leaching. The influence of thermal exposure is less significant. About 43% of the initial phenolic acids and 10% of the glycoalkaloids remain after processing. The results of the total phenolic content assay by Folin-Ciocalteu reagent are proportional to the content of phenolic compounds determined by HPLC. Steam peeling has a higher influence on glycoalkaloid losses compared to that on phenolics. The highest amounts of phenolic compounds and glycoalkaloids were found in peeling byproduct. During processing, the amount of chlorogenic acid decreased, whereas the concentration of neochlorogenic acid increased due to isomerization. The impact of the results on potato processing technology is discussed. Y1 - 2009 UR - http://pubs.acs.org/journal/jafcau U6 - https://doi.org/10.1021/Jf901066k SN - 0021-8561 ER - TY - JOUR A1 - Kawashima, Chiho A1 - Kida, Katsuya A1 - Schweigert, Florian J. A1 - Miyamoto, Akio T1 - Relationship between plasma beta-carotene concentrations during the peripartum period and ovulation in the first follicular wave postpartum in dairy cows N2 - beta-Carotene functions independently of vitamin A in the reproductive performance of dairy cows. The concentrations of beta-carotene in plasma decrease during the dry period, and reach a nadir in about the first week postpartum. This coincides with a negative energy balance, which affects the onset of the first ovulation in early postpartum cows. Thus, we hypothesised that plasma beta-carotene concentrations during the peripartum period may affect ovulation in the first follicular wave postpartum in dairy cows. The aim of the present study was to investigate changes in the profiles of plasma beta-carotene concentrations during the peripartum period in ovulatory and anovulatory cows during the first follicular wave postpartum. We used 22 multiparous Holstein cows, which were fed a total mixed ration consisting of grass, corn silage and concentrate, and collected blood samples for beta-carotene and progesterone analysis from week 3 prepartum to week 3 postpartum when the period of day 0-6 after parturition was regarded as the parturient week (week 0). The first ovulation was confirmed using the profile of plasma progesterone concentrations and colour Doppler ultrasound. Thirteen cows ovulated during the first postpartum follicular wave. Parity, the dry-off period, calving interval, mastitis episodes, and actual 305 days' milk yield during the previous lactation, and milk composition in the last month during the previous lactation in this study did not differ between ovulatory and anovulatory cows. Differences in the plasma beta-carotene profile were observed between ovulatory and anovulatory cows. Plasma P-carotene concentrations at week 3 prepartum were greater in ovulatory cows (2.97 +/- 0.24mg/L) than in anovulatory cows (1.53 +/- 0.14mg/L; P < 0.001), after that its concentrations in ovulatory cows decreased and reached the lowest level at week I postpartum, although its concentrations in anovulatory cows remained unchanged. No differences in plasma P-carotene concentrations between the two groups were observed postpartum. The present study indicates for the first time that the lower P-carotene concentrations in plasma during the prepartum period is associated with anovulation during the first follicular wave postpartum. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/03784320 U6 - https://doi.org/10.1016/j.anireprosci.2008.02.008 SN - 0378-4320 ER - TY - JOUR A1 - Hertrampf, Torsten A1 - Ledwig, C. A1 - Kulling, Sabine A1 - Molzberger, Almut A1 - Moeller, Frank J. A1 - Zierau, Oliver A1 - Vollmer, Günter A1 - Moors, Stefan A1 - Degen, Gisela H. A1 - Diel, Patrick T1 - Responses of estrogen sensitive tissues in female Wistar rats to pre- and postnatal isoflavone exposure N2 - Effects of isoflavones on estrogen sensitive tissues are discussed controversially This study was designed to investigate tissue specific effects of an isoflavone exposure through different periods of life in female Wistar rats and to compare the effects of genistein (GEN) to those of mixed dietary isoflavones, GEN and daidzein (DAI). One group received an isoflavone-free diet (IDD). another was fed an isoflavone-rich diet (IRD) and the third group an IDD supplemented with GEN (GEN(d)) prior to mating. throughout pregnancy and up to weaning The offspring were kept on the respective diets during growth. puberty and adulthood The weight of the uterus, the height of the uterine and vaginal epithelium, the bone mineral density of the tibia, and the expression of the estrogen sensitive gene CaBP9K in the liver were determined. At d21, the uterine weight, the uterine epithelium and the expression of CaBP9K in the liver were significantly stimulated in GEN(d) animals compared to IDD and IRD Interestingly, bone mineral density was increased in GEN(d) and in IRD animals Around puberty (d50) neither uterine wet weights nor trabecular bone density differed significantly among the isoflavone groups and the IDD control. At d80 nosignificant differences in uterine weight were observed among IDD. GEN(d) and IRD animals. However, bone mineral density was increased in GEN(d) and IRD animals In summary, Our results demonstrate that lifelong dietary exposure to isoflavones can affect estrogen sensitive tissues, apparently in a tissue selective manner With respect to health risk and benefit our data indicate that an increased bone mineral density can be achieved by lifelong exposure to an IRD. which, in contrast to GEN supplementation, does not seem to stimulate the proliferation of the uterine epithelium Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/03784274 U6 - https://doi.org/10.1016/j.toxlet.2009.08.019 SN - 0378-4274 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 - Griebsch, Christine A1 - Arndt, Gisela A1 - Raila, Jens A1 - Schweigert, Florian J. A1 - Kohn, Barbara T1 - C-reactive protein concentration in dogs with primary immune-mediated hemolytic anemia N2 - Background Canine primary immune-mediated hemolytic anemia (IMHA) is associated with a high-mortality rate. C- reactive protein (CRP) is the most important acute-phase protein in dogs and may have value as a marker of prognosis or response to treatment in IMHA. Objective The objectives of this study were to evaluate serum CRP concentration in dogs with primary IMHA at presentation and during treatment, to assess potential differences based on survival time, and to compare CRP with other laboratory parameters of inflammation and prognosis. Methods Inclusion criteria for primary IMHA were anemia (PCV < 0.30 L/L), a positive Coombs' test or persistent autoagglutination of erythrocytes, and the exclusion of underlying diseases by other diagnostic tests. Dogs were divided into 2 groups based on survival: dogs that were still alive 14 days after start of treatment (group 1) and dogs that died or were euthanized before day 14 (group 2). Serum CRP concentration, a CBC, and a biochemistry profile were performed on days 0, 3, 8, and 14. Serum CRP also was determined in 25 clinically healthy dogs. Results CRP concentration in the 25 clinically healthy dogs ranged from 0- 8.9 mu g/mL (median 2.2 mu g/mL). Thirty dogs were diagnosed with primary IMHA, 24 in group 1 and 6 in group 2. On day 0, CRP concentration in dogs in both groups (median 224 mu g/mL) was increased above the reference interval. In group 1 dogs, median CRP concentration was 242 mu g/mL on day 0, 69 mu g/mL on day 3, 35 mu g/mL on day 8, and 2 mu g/mL on day 14. In group 2 dogs, median CRP concentration was 194 mu g/mL on day 0, 119 mu g/mL on day 3, and 41 mu g/mL on day 8; only 1 dog in group 2 survived to day 8. There was a significant correlation between CRP and total WBC concentrations on days 0 and 3 (r=-.598, P=.003). Conclusions Serum CRP concentration was markedly increased in dogs with primary IMHA. CRP concentration did not differ based on patient survival, but might be a marker for long-term monitoring of these patients. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=0275-6382 U6 - https://doi.org/10.1111/j.1939-165X.2009.00146.x SN - 0275-6382 ER - TY - JOUR A1 - Gebhardt, Constance A1 - Hirschberger, Johannes A1 - Rau, Stefanie A1 - Arndt, Gisela A1 - Krainer, Karen A1 - Schweigert, Florian J. A1 - Brunnberg, Leo A1 - Kaspers, Bernd A1 - Kohn, Barbara T1 - Use of C-reactive protein to predict outcome in dogs with systemic inflammatory response syndrome or sepsis N2 - Background There is a high mortality rate in patients with systemic inflammatory response syndrome (SIRS) or sepsis. Therefore, an early diagnosis and prognostic assessment is important for optimal therapeutic intervention. The objective of the study was to evaluate if baseline values and changes in serum C-reactive protein (CRP) might predict survival in dogs with SIRS and sepsis. Design Prospective study; July 2004 to July 2005. Setting Small Animal Clinic, Berlin, Clinic of Small Animal Medicine, Munich. Animals Sixty-one dogs. Measurements and Main Results For the CRP analysis blood was drawn on day 0, 1, and 2; CRP was measured using a commercial ELISA test kit. Thirteen dogs suffered from nonseptic SIRS and 48 dogs from sepsis. The 14-day survival rate was 61% (69% nonseptic SIRS, 58% sepsis). Serum CRP was higher in sick dogs compared with controls (P < 0.001). Over the 3-day period surviving dogs (n=31) displayed a significantly greater decrease in CRP than nonsurvivors (n=10) (P=0.001). No correlation was found between the initial CRP concentrations and the survival rate. The changes in CRP corresponded to the survival rate (P=0.01). Conclusion There was no significant relationship between the survival rate in dogs with nonseptic SIRS or sepsis and the initial serum CRP concentrations. There was a correlation between decreasing CRP concentrations and recovery from disease. However, the changes in CRP concentrations over a 3-day period correctly predicted survival in 94% of dogs and death in 30% of the dogs (false positive rate 22%). Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=1479-3261 U6 - https://doi.org/10.1111/j.1476-4431.2009.00462.x SN - 1479-3261 ER - TY - JOUR A1 - Frey, Simone K. A1 - Spranger, Joachim A1 - Henze, Andrea A1 - Pfeiffer, Andreas F. H. A1 - Schweigert, Florian J. A1 - Raila, Jens T1 - Factors that influence retinol-binding protein 4-transthyretin interaction are not altered in overweight subjects and overweight subjects with type 2 diabetes mellitus N2 - Retinol-binding protein 4 (RBP4) is an adipokine bound in plasma to transthyretin (TTR), which prevents its glomerular filtration and subsequent catabolism in the kidney. Alterations of this interaction have been Suggested to be implicated in the elevation of RBP4 that are thought to contribute to the development Of insulin resistance associated with obesity and type 2 diabetes mellitus (T2DM). However, the factors linking RBP4 to TTR in humans are not clear. Therefore, this Study evaluated parameters influencing the RBP4-TTR interaction and their relation to obesity and T2DM. The RBP4 and TTR levels were quantified in plasma of 16 lean controls, 28 overweight controls, and 14 overweight T2DM patients by enzyme-linked immunosorbent assay. Transthyretin isoforms involved in RBP4 binding were determined by linear matrix-assisted laser desorption/ionization-time of flight-mass spectrometry after RBP4 coimmunoprecipitation. Holo-RBP4 (retinol-bound) and apo-RBP4 (retinol-free) were assessed by immunoblotting using nondenaturating polyacrylamide gel electrophoresis. Plasma levels of both RBP4 and TTR did not differ among the groups of lean controls, overweight controls, and overweight T2DM subjects. Using RBP4 immunoprecipitation, 4 mass signals were observed for TTR representing native, S-cysteinylated, S-cysteinglycinylated, and S-glutathionylated TTR. No differences in peak intensity of TTR isoforms were observed among the groups. Moreover, no differences in the ratio of holo- and apo-RBP4 were evident. The results suggest that circulating RBP4 and TTR were not affected by human obesity or T2DM, which might be attributed to the absence of alterations of TTR isoforms and the ratio of holo- and apo-RBP4 that might modify the TTR-RBP4 interaction. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/00260495 U6 - https://doi.org/10.1016/j.metabol.2009.05.003 SN - 0026-0495 ER - TY - JOUR A1 - Frey, Simone K. A1 - Henze, Andrea A1 - Nagl, Britta A1 - Raila, Jens A1 - Scholze, Alexandra A1 - Tepel, Martin A1 - Schweigert, Florian J. A1 - Zidek, Walter T1 - Effect of renal replacement therapy on retinol-binding protein 4 isoforms N2 - Background: Retinol-binding protein 4 (RBP4) levels are elevated in the serum of patients with kidney dysfunction. We recently showed that RBP4 isoforms including apo-RBP4 (RBP4 not bound to retinol) and RBP4 truncated at the C-terminus (RBP4-L, RBP4-LL) are increased in the serum of patients with kidney diseases but not in serum of patients with various liver diseases. The aim of this study was to investigate the effect of renal replacement therapy on RBP4 isoforms. Methods: We investigated serum levels of RBP4, apo-RBP4, holo-RBP4, RBP4-L, RBP4-LL, retinol and transthyretin (TTR) in 18 hemodialysis (HD) patients, 30 patients after renal transplantation (RTx) and in 35 healthy controls. RBP4 and TTR levels were measured by enzyme-linked immunosorbent assay, apo- and holo-RBP4 by native electrophoresis, retinol by high performance liquid chromatography and RBP4-L and RBP4-LL were analyzed by mass spectrometry. Results: HD and RTx patients had elevated RBP4, apo-RBP4 and RBP4-LL levels compared to controls. RTx patients had elevated amounts of RBP4-L compared to controls and elevated RBP4 and apo-RBP4 levels compared to HD patients. Conclusion: The results demonstrate a strong correlation between kidney function and RBP4 isoforms and provide data for investigating the relation of RBP4 and insulin resistance in these patients. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/00098981 U6 - https://doi.org/10.1016/j.cca.2008.11.008 SN - 0009-8981 ER - TY - JOUR A1 - Barth, Christian A. T1 - Nutritional value of rapeseed oil and its high oleic/low linolenic variety : a call for differentiation N2 - To offer the best choice of healthy and acceptable food to the consumer a coordination of plant breeding, food processing and nutrition science is required. Here the nutritional aspects of the high oleic/low linolenic (HOLLi) varieties of rapeseed with a low alpha-linolenic acid content of about 3% are reviewed. The content of alpha-linolenic acid amounting to around 9% is the hallmark of the positive nutritional value of the original (erucic acid free) 00 varieties of rapeseed oil ("canola" quality in North America). n-3 fatty acids are endowed with the property to protect the cardiovascular system from chronic disease and the consumption of food containing n-3 fatty acids is explicitly recommended by national and international nutritional and medical authorities. Although the use of HOLLi with a low n-3 fatty acid content can be unavoidable for specific purposes, because of technological and health considerations the continuous future consumption of the original rapeseed oil with around 9% of alpha-linolenic acid by the consumer should have high priority from the standpoint of public health. To pursue this aim confusion of the consumer must be avoided by creating a new name and a new brand for HOLLi varieties. Y1 - 2009 UR - http://onlinelibrary.wiley.com/journal/10.1002/%28ISSN%291438-9312 U6 - https://doi.org/10.1002/ejlt.200900019 SN - 1438-7697 ER - TY - JOUR A1 - Bachmann, Lutz A1 - Homeier, Timo A1 - Arlt, Sebastian A1 - Brueckner, Monika A1 - Rawel, Harshadrai Manilal A1 - Deiner, Carolin A1 - Hartmann, Helmut T1 - Influence of different oral rehydration solutions on abomasal conditions and the acid-base status of suckling calves N2 - The aim of the study was to investigate the influence of oral rehydration solutions (ORS) on milk clotting, abomasal pH, electrolyte concentrations, and osmolality, as well as on the acid-base status in blood of suckling calves, as treatment with ORS is the most common therapy of diarrhea in calves to correct dehydration and metabolic acidosis. Oral rehydration solutions are suspected to inhibit abomasal clotting of milk; however, it is recommended to continue feeding cow's milk or milk replacer (MR) to diarrheic calves to prevent body weight losses. Three calves with abomasal cannulas were fed MR, MR-ORS mixtures, or water-ORS mixtures, respectively. Samples of abomasal fluid were taken before and after feeding at various time points, and pH, electrolyte concentrations, and osmolality were measured. The interference of ORS with milk clotting was examined in vivo and in vitro. To evaluate the effects of ORS on systemic acid-base status, the Stewart variables strong ion difference ([SID]), acid total ([A(tot)]), and partial pressure of CO2 (pCO(2)) were quantified in venous blood samples drawn before and after feeding. Calves reached higher abomasal pH values when fed with MR-ORS mixtures than when fed MR. Preprandial pH values were re-established after 4 to 6 h. Oral rehydration solutions prepared in water increased the abomasal fluid pH only for 1 to 2 h. Oral rehydration solutions with high [SID3] ([Na+] + [K+] - [Cl-]) values produced significantly higher abomasal pH values and area under the curve data of the pH time course. Caseinomacropeptide, an indicator of successful enzymatic milk clotting, could be identified in every sample of abomasal fluid after feeding MR-ORS mixtures. The MR-ORS mixtures with [SID3] values >= 92 mmol/L increased serum [SID3] but did not change venous blood pH. Oral rehydration solutions do not interfere with milk clotting in the abomasum and can, therefore, be administered with milk. In this study, MR-ORS mixtures with high [SID3] values caused an increase of serum [SID3] in healthy suckling calves and may be an effective treatment for metabolic acidosis in calves suffering from diarrhea. Y1 - 2009 UR - http://www.journalofdairyscience.org/ U6 - https://doi.org/10.3168/jds.2008-1487 SN - 0022-0302 ER - TY - THES A1 - Kucia, Marzena T1 - Impact of a high protein diet on maternal health status, milk composition and rearing success during pregnancy and lactation of two mouse genotypes Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Müller, Carsten T1 - Nachweis antioxidativer und chemopräventiver Eigenschaften von Naturstoffen zur Verwendung als potenzielle Nahrungsergänzungsmittel Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Hessel, Stefanie T1 - Mechanismen zur Detoxifizierung von Benzo[a]pyren-Metaboliten in der gastrointestinalen Barriere Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Hanisch, Christiana T1 - Die Wirkung eines Symbiotikums auf die Zusammensetzung der intestinalen Mikrobiota, die fäkale Exkretion von kurzkettigen Fettsäuren sowie ausgewählte Parameter der Darmfunktion bei über 65-jährigen Frauen und Männern Y1 - 2009 ER - TY - THES A1 - Henze, Andrea T1 - Chronic kidney disease and type 2 diabetes mellitus as factors influencing retinol-binding protein 4 Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Vogel, Heike T1 - Identifizierung, Charakterisierung und Eingrenzung eines Suszeptibilitätslocus (Nob3) für Adipositas und Hyperglykämie im Mausgenom Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Chadt, Alexandra T1 - Functional characterization of a novel candidate gene for obesity, Tbc 1d1 Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Brockhoff, Anne T1 - Struktur-Funktionsbeziehungen menschlicher Bitterrezeptoren Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Rade-Kukic, Koraljka T1 - Interactionof ß-lactoglobulin with sinigin and allyl-isothiocyanate - effect on protein's physico- chemical and functional properties Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Gärtner, Sonja T1 - Elektrochemische Charakterisierung von Anthocyanen und mögliche Wechselwirkungen mit biologisch relevanten Systemen Y1 - 2009 CY - Potsdam ER - TY - THES A1 - Drewes, Gunnar T1 - Die Bedeutung Eisen-Schwefel-Cluster-assoziierter Mechanismen für Mutagenese und Kanzerogenese Y1 - 2009 CY - Potsdam ER - TY - JOUR A1 - Barth, Christian A. T1 - Ein Vitamin mit zwei Gesichtern : Folat in der Vorsorge Y1 - 2009 UR - http://www.dlr-online.de/ SN - 0012-0413 ER - TY - GEN A1 - Scholtka, Bettina A1 - Schneider, Mandy A1 - Melcher, Ralph A1 - Katzenberger, Tiemo A1 - Friedrich, Daniela A1 - Berghof-Jäger, Kornelia A1 - Scheppach, Wolfgang A1 - Steinberg, Pablo T1 - A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in 80% of early (UICC I) colon cancer stages in humans N2 - Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 120 KW - Colorectal carcinomas KW - K-RAS KW - Microsatellite instability KW - Oncogenes KW - Tumour suppressor genes Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-44587 ER - TY - GEN A1 - Scholtka, Bettina A1 - Kühnel, Dana A1 - Taugner, Felicitas A1 - Steinberg, Pablo T1 - Inflammation does not precede or accompany the induction of perneoplastic lesions in the colon of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-fed rats N2 - Heterocyclic aromatic amines (HCAs) are formed in meat cooked at high temperatures for a long time or over an open flame. In this context 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant HCA in cooked meat, has been suggested to be involved in colon and prostate carcinogenesis. In the latter case it has been reported that: (1) roughly 50% of Fischer F344 male rats treated with PhIP develop carcinomas in the ventral prostate lobe at 1 year of age; (2) inflammation precedes prostatic intraepithelial neoplasia in PhIP-fed rats; (3) inflammation specifically occurs in the ventral prostate lobe of PhIP-fed rats. To test whether PhIP by itself leads to inflammation in the colon and whether a human-relevant concentration of PhIP is able to induce preneoplastic lesions in the colon, male F344 rats were fed 0.1 or 100 ppm PhIP for up to 10 months and thereafter the colon tissue was analyzed histochemically. In none of the experimental groups signs of acute or chronic colonic inflammation were observed. 0.1 ppm PhIP leads to the development of hyperplastic and dysplastic lesions in the colon of single animals, but the incidence of these lesions does not reach a statistical significance. In contrast, in rats fed 100 ppm PhIP for 10 months hyperplastic and dysplastic colonic lesions were induced in a statistically significant number of animals. It is concluded that: (1) the induction of preneoplastic lesions in rat colon by PhIP is not preceded or accompanied by an inflammatory process; (2) a human-relevant concentration of PhIP alone is not sufficient to initiate colon carcinogenesis in rats. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 119 KW - Colorectal cancer KW - Heterocyclic aromatic amines KW - Inflammation Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-44570 ER - TY - THES A1 - Wend, Korinna T1 - Konstruktion und toxikologische Nutzung von transgenen Mäusen mit den allelischen Varianten von humanen SULT1A-Genen T1 - Construction and characterisation of transgenic mice for human sulfotransferases with polymorphic SULT1A genes N2 - Eine besondere Rolle im Fremdstoffmetabolismus hat die SULT1A1 beim Menschen aufgrund der hohen Expression und breiten Gewebeverteilung. Während die humane SULT1A1 in sehr vielen Geweben exprimiert wird, wurde die murine SULT1A1 vor allem in der Leber, Lunge und Colon gefunden. Neben der Gewebeverteilung spielt auch der Polymorphismus im humanen SULT1A1-Gen eine bedeutende Rolle. Der häufigste Polymorphismus in diesem Gen führt zu einer Aminosäuresubstitution von Arginin zu Histidin an Position 213. Die Genvariante mit Histidin (auch als SULT1A1*2 bezeichnet) codiert für ein Protein mit einer geringen Enzymaktivität und einer reduzierten Enzymmenge in Thrombocyten. Über den Einfluss dieser allelischen Varianten in anderen Geweben ist bislang wenig bekannt. In vorausgegangenen epidemiologischen Studien wurden mögliche Korrelationen zwischen den Genvarianten und der Krebsentstehung in verschiedenen Geweben untersucht. Diese Daten liefern jedoch widersprüchliche Ergebnisse zum Krebsrisiko. Aufgrund der strittigen epidemiologischen Daten sollten Tiermodelle generiert werden, um die häufigsten SULT1A1-Allele hinsichtlich der Empfindlichkeit gegenüber Nahrungs- und Umweltkanzerogenen zu untersuchen. Zur Erzeugung transgener (tg) Mauslinien wurde mittels Mikroinjektion der codierenden Genbereich und große flankierende Humansequenzen stromaufwärts und stromabwärts in das Mausgenom integriert. Es wurden mehrere Mauslinien hergestellt. Zwei davon, die Mauslinie 31 mit dem SULT1A1*1-Allel und die Mauslinie 28 mit dem SULT1A1*2-Allel, wurden eingehend analysiert. In beiden Linien wurde eine identische Kopienzahl des Transgens ermittelt. Proteinbiochemische Charakterisierungen zeigten eine weitgehend dem Menschen entsprechende Gewebeverteilung und zelluläre und subzelluläre Lokalisation der humanen SULT1A1 in der Linie (Li) 28. In Li 31 wurden Unterschiede zu Li 28 sowohl in der Gewebeverteilung als auch in der zellulären Lokalisation des exprimierten humanen Proteins ermittelt. Dabei war die Expression auf Proteinebene in der SULT1A1*2-tg Linie generell stärker als in der SULT1A1*1-Linie. Dieses Ergebnis war überraschend, denn in humanen Thrombocyten führt das SULT1A1*1-Allel zu einem höheren Gehalt an SULT1A1-Protein als das SULT1A1*2-Allel. Zur Analyse der unterschiedlichen Proteinexpressionen in den tg Mauslinien wurde die cDNA und der 5´-flankierende Bereich des SULT1A1-Gens sequenziert. In beiden tg Linien entsprach die Sequenz der cDNA der Referenzsequenz aus der Gendatenbank (Pubmed). In der 5´-flankierenden Region wurden bekannte Polymorphismen analysiert und unterschiedliche Haplotypen in den tg Linien an den Positionen -624 und -396 ermittelt. Dabei wurde in der Li 31 der Haplotyp detektiert, der in der Literatur mit einer höheren SULT1A1-Enzymaktivität beschrieben wird. Der mögliche Zusammenhang zwischen Transkriptionsrate und Proteinexpression wurde in RNA-Expressionsanalysen im codierenden und 5´-nicht codierenden Bereich (mit den alternativen Exons 1B und 1A) untersucht. Im codierenden Bereich und im Exon 1B konnte in den untersuchten Organen eine höhere RNA-Expression in der Li 28 im Vergleich zur Li 31 ermittelt werden. Außer in der Lunge wurde für Exon 1B eine identische RNA-Expression detektiert. RNA, die Exon 1A enthielt, wurde in allen untersuchten Organen der Li 28, aber nur in der Lunge bei der Li 31 gefunden. In beiden tg Linien konnten mit den Exon 1A-Primern jedoch auch größere PCR-Produkte ermittelt werden. Dieser Unterschied im Exon 1A und mögliche Spleißvarianten könnten damit für die unterschiedliche Proteinexpression des humanen SULT1A1-Proteins in den beiden tg Mauslinien sein. Die in dieser Arbeit generierten und charakterisierten tg Mausmodelle wurden in einer toxikologischen Studie eingesetzt. Es wurde das heterozyklische aromatische Amin 2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridin (PhIP) verwendet. PhIP wird beim Erhitzen und Braten von Fleisch und Fisch gebildet und könnte mit der erhöhten Krebsentstehung im Colon in der westlichen Welt im Zusammenhang stehen. Mittels 32P-Postlabelling sollte der Einfluss der zusätzlichen Expression der humanen SULT-Proteine auf die PhIP-DNA-Adduktbildung analysiert werden. Dabei wurden mehr DNA-Addukte in den tg Tieren als in den Wildtyp-Mäusen ermittelt. Die Konzentration der gebildeten DNA-Addukte korrelierte mit der Expressionsstärke des humanen SULT1A1-Proteins in den tg Mäusen. An den in dieser Arbeit generierten tg Mauslinien mit den häufigsten allelischen Varianten des SULT1A1-Gens konnten Unterschiede auf RNA- und Protein-Ebene ermittelt werden. Zudem konnte gezeigt werden, dass die Expression der humanen SULT1A1 eine Auswirkung sowohl auf die Stärke als auch das Zielgewebe der DNA-Adduktbildung in vivo hat. N2 - In humans, SULT1A1 and its polymorphic variants play an important role in xenobiotic metabolism and display a broad tissue distribution and high expression level. This enzyme is expressed in almost every human organ whereas in mice SULT1A1 can only be detected in liver, lung and colon. The most common polymorphism of this gene leads to an amino acid substitution from arginine to histidine at the position 213. In platelets, the allele encoding histidine (also designated as SULT1A1*2) is associated with both low activity and low thermal stability of the SULT protein. However, so far only little is known about the significance of these allelic variants in the other tissues with hSULT1A1 expression. Previous epidemiological studies have made attempts to correlate SULT1A1 allelic variants and cancer development, their data, however, have been contradictory for an appropriate cancer risk assessment. In this thesis, we addressed the effect of the hSULT1A1 genetic variability on the susceptibility to nutritional and environmental carcinogens using transgenic (tg) mouse models. We generated tg mice carrying the most common allelic variants of the human SULT1A1 gene. The coding region and large flanking human sequences upstream and downstream of the hSULT1A1 gene were integrated randomly into the mouse genome by microinjection. Several tg mouse lines were generated. Two of them, line (li) 31 with the SULT1A1*1 allele and li 28 with the SULT1A1*2 allele, were analysed in detail. At first, an identical transgene copy number was detected in both lines. Furthermore, biochemical characterization of li 28 showed that the tissue distribution, the cellular and subcellular localisation of the protein were very similar to those in humans. In contrast, li 31 exhibited differences in tissue distribution and cellular localisation of the human protein compared to li 28. The protein expression level in the tg line with SULT1A1*2 (li 28) was generally higher than in SULT1A1*1 (li 31) mice. These results were surprising since the SULT1A1*1 allele in human platelets usually leads to a higher amount of SULT1A1 protein compared to the SULT1A1*2 allele. To investigate these differences, we sequenced the cDNA and 5´-flanking region of the SULT1A1 gene. In both tg mouse lines, the cDNA sequence was identical to the reference sequence from the gene databank (Pubmed). We subsequently analysed the common polymorphisms of the 5´-flanking region, and determined different haplotypes at position -624 and -396 in the tg mouse lines. According to the literature, the haplotype associated with a higher SULT1A1 enzyme activity, we detected in li 31. We analyzed the possible correlation between gene transcription and protein expression by measuring RNA expression levels of the coding and the non-coding region (with alternative exons 1B and 1A). We detected a higher RNA expression level of the coding region and exon 1B in li 28 compared to li 31, whereas RNA for exon 1A was only found in li 28 in all investigated tissues, but only in lung in li 31. Furthermore we detected with exon 1A-primers larger RNA in both lines. These differences in exon 1A expression accompanied by potential splicing variants could be responsible for the different expression and activity of the human SULT1A1 protein in both tg mouse lines. In order to validate our generated and characterized tg mouse models as toxicological in vivo models, we used them for the evaluation of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP). PhIP is typically generated during heating and roasting of meat and fish and is suggested to be associated with an increased colon cancer incidence in the western world. We measured the impact of the additionally expressed human SULT proteins on the PhIP-DNA adduct level by 32P-postlabelling. We detected significantly higher DNA adduct levels in tg compared to wildtype mice, which correlated positively with the expression pattern of the human SULT1A1 protein in the tg mice. In conclusion, in this thesis, we have successfully generated and validated the transgenic mouse lines carrying the most common allelic variants of the human SULT1A1 gene. Interestingly, these lines exhibited differences in both the SULT1A1 RNA and protein levels. Using these transgenic mouse models as in vivo toxicological tools we have shown that the expression of human SULT1A1 in mice has a decisive impact on the strength and the target tissue of DNA adducts. KW - transgenes Mausmodell KW - Polymorphismus KW - Sulfotransferase KW - SULT1A1 KW - SULT1A2 KW - PhIP KW - heterocyclisches aromatisches Amin KW - transgenic mousemodel KW - polymorphism KW - sulfotransferase KW - SULT1A1 KW - SULT1A2 KW - PhIP KW - heterocyclic aromatic amine Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-42052 ER - TY - THES A1 - Behrens, Verena T1 - Die Rolle des Glucosetransporters 8 (Slc2a8) in der Regulation der Glucosehomöostase, der Spermienmotilität sowie des Verhaltens T1 - The physiological role of glucose transporter 8 (Slc2a8) in regulation of glucose homeostasis, sperm motility and behavior N2 - Der ubiquitär exprimierte, multifunktionale Glucosetransporter GLUT8 gehört zur Klasse III der Familie der passiven Glucosetransporter, die aus insgesamt 14 Proteinen besteht. Die fünf Mitglieder der Klasse IIII unterscheiden sich strukturell leicht von den Mitgliedern der Klasse I und II (Joost und Thorens, 2001). GLUT8 besitzt ein N-terminales Dileucin-Motiv, das Teil eines [DE]XXXL[LI] Motivs ist, welches für die Sortierung des Transporters in späte Endosomen und Lysosomen verantwortlich ist (Augustin et al., 2005). Da bis heute kein Signal identifiziert wurde, das eine Translokation des Transporters zur Plasmamembran auslöst, wird eine intrazelluläre Funktion von GLUT8 vermutet (Widmer et al., 2005). Im Rahmen der vorliegenden Arbeit wurde die intrazelluläre Funktion des Transporters in der Regulation der Glucosehomöostase des Körpers durch Analyse einer Slc2a8-knockout-Maus untersucht. Die homozygote Deletion des Transporters erbrachte lebensfähige Nachkommen, die sich augenscheinlich nicht von ihren Wildtyp-Geschwistern unterschieden. Allerdings wurde bei Verpaarungen heterozygoter Mäuse eine verminderte Anzahl an Slc2a8-/--Nachkommen beobachtet, die signifikant von der erwarteten Mendel’schen Verteilung abwich. Da Slc2a8 die höchste mRNA-Expression in den Testes aufwies und die Überprüfung der Fertilität mittels verschiedener homozygoter Verpaarungen eine Störung der weiblichen Fortpflanzungsfähigkeit ausschloss, wurden die Spermatozoen der Slc2a8-/--Mäuse eingehender untersucht. Als Ursache für die verringerte Anzahl von Slc2a8-/--Geburten wurde eine verminderte Prozentzahl motiler Slc2a8-/--Spermien ermittelt, die durch eine unzureichende mitochondriale Kondensation in den Spermien bedingt war. Diese Veränderung war mit einem reduzierten mitochondrialen Membranpotential assoziiert, was eine verminderte ATP-Produktion nach sich zog. Somit scheint GLUT8 in den Spermien an einem intrazellulären Transportprozess beteiligt zu sein, der einen Einfluss auf die oxidative Phosphorylierung der Mitochondrien ausübt. Im Gehirn wurde Slc2a8 besonders stark im Hippocampus exprimiert, der in der Regulation von körperlicher Aktivität, Explorationsverhalten, Erinnerungs- und Lernprozessen sowie Angst- und Stressreaktionen eine Rolle spielt. Außerdem wurde GLUT8 im Hypothalamus nachgewiesen, der unter anderem an der Regulation der Nahrungsaufnahme beteiligt ist. Die Slc2a8-/--Mäuse zeigten im Vergleich zu ihren Slc2a8+/+-Geschwistern eine signifikant gesteigerte körperliche Aktivität, die zusammen mit der von Membrez et al. (2006) publizierten erhöhten Zellproliferation im Hippocampus auf eine Nährstoffunterversorgung dieses Areals hindeutet. Die Nahrungsaufnahme war in Abwesenheit von GLUT8 nicht verändert, was zusammen mit dem nur geringfügig niedrigeren Körpergewicht der Slc2a8-/--Mäuse eine Funktion von GLUT8 im Glucose-sensing der Glucose-sensitiven Neurone des Gehirns ausschließt. Das leicht reduzierte Körpergewicht der Slc2a8-/--Mäuse ließ sich keinem bestimmten Organ- oder Gewebetyp zuordnen, sondern schien durch eine marginale Gewichtsreduktion aller untersuchten Gewebe bedingt zu sein. Zusammen mit den erniedrigten Blutglucosespiegeln und der anscheinend gesteigerten Lebenserwartung zeigten die Slc2a8-/--Mäuse Symptome einer leichten Nährstoffunterversorgung. GLUT8 scheint daher am Transport von Zuckerderivaten, die während des lysosomalen/endosomalen Abbaus von Glykoproteinen anfallen, beteiligt zu sein. Die so wiederaufbereiteten Zucker dienen dem Körper offenbar als zusätzliche Energiequelle. N2 - The family of facilitative glucose transporters consists of 14 different members in human, which are divided into three classes (Joost and Thorens, 2001). The class III family member GLUT8 contains an amino-terminal dileucine sorting signal, which is part of the highly conserved [DE]XXXL[LI] motif responsible for the localization of GLUT8 in lysosomes and late endosomes (Augustin et al., 2005). To date there is no stimulus known, which translocates the transporter to the plasma membrane, therefore an intracellular function rather than at the cell surface is considered (Widmer et al., 2005). The aim of the present dissertation was to analyze the intracellular role of GLUT8 in the regulation of whole body glucose homeostasis, by the characterization of the corresponding knockout mice (Slc2a8-/-). Slc2a8-/- mice were viable and showed no obvious disparity to their wild-type littermates. However, analysis of the offspring distribution of heterozygous mating provided a reduced number of born Slc2a8-/- offspring which differed significantly from the expected Mendelian distribution. Because Slc2a8 mRNA is expressed at highest levels in the testis and the female Slc2a8-/- mice showed no alterations in fertility, we further investigated the function of Slc2a8-/- spermatozoa. An impaired mitochondrial condensation in the Slc2a8-/- spermatozoa, which was associated with decreased ATP levels resulted in a reduced number of motile Slc2a8-/- sperm, which appeared to be responsible for the reduced number of born Slc2a8-/- offspring. Therefore in sperm cells GLUT8 seems to be important for an intracellular transport process, which exerts an influence on the oxidative phosphorylation in the mitochondria. In the brain Slc2a8 is expressed at highest levels in the hippocampus, which is important for the regulation of physical activity, exploration behaviour, memory and learning as well as anxiety related behaviour. Additionally, GLUT8 was detected in the hypothalamus, which is amongst others involved in the regulation of food intake. The Slc2a8-/- mice showed a significant increase in locomotor activity, which indicates a moderate undersupply of the hippocampus area. According to this finding the group of Membrez et al. (2006) observed a raised cell proliferation in the hippocampus of Slc2a8-/- mice. The fact that no alterations in food intake and only a moderate reduction in body weight was detected in Slc2a8-/- mice, indicates that GLUT8 is not important for the hypothalamic glucose sensing. The marginal decreased body weight of the Slc2a8-/- mice appeared to be associated with a slightly reduced weight of different tissues. Together with the lowered blood glucose concentrations and the apparently enhanced lifespan, the Slc2a8-/- mice showed symptoms of a moderate undersupply compareable to caloric restriction. Thus, we hypothesize that GLUT8 is important for the transport of sugar derivatives which arise during lysosomal/endosomal degradation of glycoproteins. These recycled sugars may serve as an additional energy source in the cell. KW - Glucosetransport KW - GLUT8 KW - Spermienmotilität KW - Verhalten KW - Glucosehomöostase KW - glucose transport KW - GLUT8 KW - sperm motility KW - behavior KW - glucose homeostasis Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-36308 ER - TY - THES A1 - Nell, Sandra T1 - Vitamin E und der vesikuläre Transport : Untersuchungen zu den genregulatorischen Funktionen von Vitamin E mittels Microarray- und real time PCR-Analysen in der Maus und funktionellen in vitro Assays in RBL-2H3 Zellen T1 - Vitamin E and the vesicular transport : examination of the generegulatory functions of vitamin E using microarrays and real time PCR analyses in the mouse and functional in vitro assays in RBL-2H3 cells N2 - Vitamin E wird immer noch als das wichtigste lipophile Antioxidanz in biologischen Membranen betrachtet. In den letzten Jahren hat sich jedoch der Schwerpunkt der Vitamin E-Forschung hin zu den nicht-antioxidativen Funktionen verlagert. Besonderes Interesse gilt dabei dem α-Tocopherol, der häufigsten Vitamin E-Form im Gewebe von Säugetieren, und seiner Rolle bei der Regulation der Genexpression. Das Ziel dieser Dissertation war die Untersuchung der genregulatorischen Funktionen von α-Tocoperol und die Identifizierung α-Tocopherol-sensitiver Gene in vivo. Zu diesem Zweck wurden Mäuse mit verschiedenen Mengen α-Tocopherol gefüttert. Die Analyse der hepatischen Genexpression mit Hilfe von DNA-Microarrays identifizierte 387 α-Tocopherol-sensitive Gene. Funktionelle Clusteranalysen der differentiell exprimierten Gene zeigten einen Einfluss von α-Tocooherol auf zelluläre Transportprozesse. Besonders solche Gene, die an vesikulären Transportvorgängen beteiligt sind, wurden größtenteils durch α-Tocopherol hochreguliert. Für Syntaxin 1C, Vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and Syntaxin binding protein 1 konnte eine erhöhte Expression mittels real time PCR bestätigt werden. Ein funktioneller Einfluss von α-Tocopherol auf vesikuläre Transportprozesse konnte mit Hilfe des in vitro β-Hexosaminidase Assays in der sekretorischen Mastzelllinie RBL-2H3 gezeigt werden. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer konzentrationsabhängigen Erhöhung der PMA/Ionomycin-stimulierten Sekretion der β-Hexosaminidase. Eine erhöhte Expression ausgewählter Gene, die an der Degranulation beteiligt sind, konnte nicht beobachtet werden. Damit schien ein direkter genregulatorischer Effekt von α-Tocopherol eher unwahrscheinlich. Da eine erhöhte Sekretion auch mit β-Tocopherol aber nicht mit Trolox, einem hydrophilen Vitamin E-Analogon, gefunden wurde, wurde vermutet, dass α-Tocopherol die Degranulation möglicherweise durch seine membranständige Lokalisation beeinflussen könnte. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer veränderten Verteilung des Gangliosids GM1, einem Lipid raft Marker. Es wird angenommen, dass diese Membranmikrodomänen als Plattformen für Signaltransduktionsvorgänge fungieren. Ein möglicher Einfluss von Vitamin E auf die Rekrutierung/Translokation von Signalproteinen in Membranmikrodomänen könnte die beobachteten Effekte erklären. Eine Rolle von α-Tocopherol im vesikulären Transport könnte nicht nur seine eigene Absorption und seinen Transport beeinflussen, sondern auch eine Erklärung für die bei schwerer Vitamin E-Defizienz auftretenden neuronalen Dysfunktionen bieten. Im zweiten Teil der Arbeit wurde die α-Tocopheroltransferprotein (Ttpa) Knockout-Maus als genetisches Modell für Vitamin E-Defizienz verwendet, um den Effekt von Ttpa auf die Genexpression und die Gewebeverteilung von α-Tocopherol zu analysieren. Ttpa ist ein cytosolisches Protein, das für die selektive Retention von α-Tocopherol in der Leber verantwortlich ist. Die Ttpa-Defizienz resultierte in sehr geringen α-Tocopherol-Konzentrationen im Plasma und den extrahepatischen Geweben. Die Analyse der α-Tocopherol-Gehalte im Gehirn wies auf eine Rolle von Ttpa bei der α-Tocopherol-Aufnahme ins Gehirn hin. N2 - Vitamin E is still considered the most important lipid-soluble antioxidant within biological membranes. However, in the last years the non-antioxidant functions of vitamin E have become the focus of vitamin E research. From the eight members of the vitamin E family, specific emphasis is given to α-tocopherol, the most abundant vitamin E form in mammalian tissues, and its role in the regulation of gene expression. The aim of this thesis was the analysis of the gene regulatory functions of α-tocopherol and the identification of α-tocopherol sensitive genes in vivo. For this purpose mice were fed diets differing in α-tocopherol content. The analysis of hepatic gene expression using DNA microarrays identified 387 α-tocopherol-sensitive genes. Functional cluster analyses of these differentially expressed genes demonstrated an influence of α-tocopherol on cellular transport processes. Especially the expression of genes involved in vesicular trafficking was largely upregulated by α-tocopherol. Upregulation of syntaxin 1C, vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and syntaxin binding protein 1 was verified by real time PCR. A role of α-tocopherol in exocytosis was shown by the in vitro β-hexosaminidase release assay in the secretory mast cell line RBL-2H3. Incubation with α-tocopherol resulted in a concentration dependent increase of PMA/ionomycin-stimulated secretion of β-hexosaminidase. Induction of selected genes involved in degranulation was not observed at any time point. Thus, a direct gene-regulatory effect of α-tocopherol seemed rather unlikely. Since increased secretion was also observed with ß-tocopherol but not with trolox, a water-soluble analog of vitamin E, it was hypothesized that α-tocopherol might affect degranulation through its localization at the plasma membrane. Incubation of cells with α-tocopherol changed the distribution of the gangliosid GM1, a Lipid raft marker. These membrane microdomains are assumed to function as signaling platforms. An possible influence of vitamin E on the recruitment/translocation of signaling proteins into membrane microdomains could explain the observed effects. A role of α-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe α-tocopherol deficiency. In the second part of this dissertation the α-tocopherol transfer protein (Ttpa) knockout-mouse as a model of genetic vitamin E deficiency was used to analyze the effect of Ttpa gene expression and tissue distribution of α-tocopherol. Ttpa is a cytosolic protein, which is responsible for the selective retention of α-tocopherol in the liver. Its deficiency resulted in very low α-tocopherol concentrations in plasma and extrahepatic tissues. Analysis of α-tocopherol contents in brain indicated a role for Ttpa in the uptake of α-tocopherol into the brain. KW - Vitamin E KW - Microarray KW - Genregulation KW - vesikulärer Transport KW - α-Tocopheroltransferprotein (Ttpa) KW - vitamin E KW - microarray KW - gene regulation KW - vesicular transport KW - alpha-tocopherol transfer protein (Ttpa) Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-35710 ER - TY - THES A1 - Schmidt, Antje T1 - Untersuchung des Recyclings Kaede-fusionierter Corticotropin-Releasing-Factor Rezeptoren Typ 1 T1 - Use of Kaede-Fusions to Visualize Recycling of the Corticotropin-Releasing Factor Receptor Type 1 N2 - Aktivierte G-Protein-gekoppelte Rezeptoren (GPCR) werden schnell desensitisiert, internalisiert und anschließend entweder lysosomal degradiert oder zur Plasmamembran (PM) recycelt. Zur Resensitisierung der Zellen tragen neben recycelten auch neusynthetisierte Rezeptoren bei. Die Überlagerung beider Prozesse erschwert die Untersuchung des Rezeptorrecyclings. In dieser Arbeit sollte mit Hilfe des photokonvertierbaren Fluoreszenzproteins Kaede eine Technik entwickelt werden, mit der es möglich ist Recycling- von Neusyntheseprozessen zu trennen und das Recycling von GPCR mikroskopisch in Echtzeit zu beobachten. Als Modellproteine wurden der Vasopressin-1a-Rezeptor V1aR (recycelnder Rezeptor), der Vasopressin-2-Rezeptor V2R (degradierter Rezeptor) und der Corticotropin-Releasing Factor-Rezeptor Typ 1 (CRF1R) verwendet, wobei bei Letzterem untersucht werden sollte, ob er nach Stimulation zur PM zurücktransportiert wird. Da Kaede als fluoreszierendes Protein mit den GPCR fusioniert wird, wurde zunächst überprüft, ob es die Eigenschaften der Rezeptoren verändert und generell für Transportstudien geeignet ist. Eventuell könnte die bereits publizierte Tetramerisierung von Kaede seine Anwendung verhindern oder erschweren. Mittels Fluoreszenz-Korrelationsspektroskopie konnte gezeigt werden, dass Kaede nicht tetramerisiert, wenn es an ein Membranprotein fusioniert ist. Außerdem konnte in in vitro- und Zellkulturexperimenten belegt werden, dass die native und die photokonvertierte Form von Kaede gleichermaßen stabil sind. Darüber hinaus zeigten Kaede-fusionierte GPCR sowohl in Kolokalisationsstudien als auch in Agonistbindungs- und Rezeptoraktivierungsexperimenten die gleichen Eigenschaften wie CFP- bzw. die unfusionierte Rezeptoren. Lediglich die Expression der Kaede-fusionierten Rezeptoren war geringer. Parallel wurde anhand der bereits publizierten Kaede-Struktur versucht, die Tetramerisierung des Proteins durch den Austausch interagierender Aminosäuren zu unterbinden. Die eingeführten Mutationen bewirkten aber eine Fehlfaltung des Proteins und damit den Verlust der Fluoreszenz. Da zuvor gezeigt werden konnte, dass Kaede-fusionierte Membranproteine nicht tetramerisieren und nicht die Eigenschaften der fusionierten Proteine verändern, war monomerisiertes Kaede zur Untersuchung des Rezeptorrecyclings nicht notwendig. Im zweiten Teil der Arbeit wurde mit Hilfe von Kaede-Fusionsproteinen und mikroskopischer Testsysteme das noch unbekannte Recyclingverhalten des CRF1R untersucht. Hierfür wurden die Kaede-fusionierten Rezeptoren in eukaryotischen Zellen exprimiert und mit Agonisten internalisiert. Die internalisierten Rezeptoren wurden in Endosomen selektiv mit UV-Strahlung photokonvertiert. Anschließend wurde der Transport der photokonvertierten Form verfolgt. Sowohl beim CRF1R als auch beim V1aR wurden Signale in der PM detektiert, beim V2R hingegen nicht. Dies zeigt, dass es sich beim CRF1R um einen recycelnden Rezeptor handelt. Die als Kontrolle eingesetzten Rezeptoren verhielten sich in diesem Experiment wie erwartet: Der V1aR wurde zur PM zurücktransportiert, der V2R nicht. Diese Ergebnisse konnten mit Hilfe biochemischer und durchflusscytometrischer Experimente bestätigt werden. Die Internalisierung des CRF1R verläuft Clathrin-vermittelt in Anwesenheit von β-Arrestin. Je nach Stabilität der β Arrestin-Interaktion unterscheidet man zwei Klassen von Rezeptoren: Klasse A-Rezeptoren interagieren transient mit β Arrestin und können recyceln. Im Gegensatz dazu gehen Klasse B-Rezeptoren eine stabile Interaktion mit β Arrestin ein und werden nach Internalisierung degradiert. In mikroskopischen Untersuchungen konnte für die aktivierten CRF1R und V1aR eine Rekrutierung von β Arrestin zur PM und eine transiente Interaktion mit β Arrestin gezeigt werden (Klasse A-Rezeptoren). Für den V2R wurde dagegen eine stabile Interaktion mit β Arrestin beobachtet (Klasse B-Rezeptor). Diese Daten stützen die Ergebnisse des Kaede-basierten Recyclingversuchs und zeigen, dass der CRF1R ein recycelnder Rezeptor ist. Ferner wurde untersucht, ob der CRF1R zu den schnell oder langsam recycelnden Rezeptoren zählt. Schnell recycelnde Rezeptoren werden direkt aus frühen Endosomen, langsam recycelnde hingegen über das Trans-Golgi-Netzwerk (TGN) bzw. über Recycling-Endosomen zur PM transportiert. Als Marker für das TGN oder die Recycling-Endosomen wurde Rab11 verwendet. In Kolokalisationsstudien konnte gezeigt werden, dass der CRF1R den langsam recycelnden Rezeptoren zugeordnet werden kann. Zusammenfassend konnte in dieser Arbeit belegt werden, dass Kaede als Fusionspartner für Membranproteine genutzt werden kann um deren Transport in Echtzeit zu studieren. Damit wurde erstmals eine mikroskopische Methode etabliert, die es erlaubt recycelnde von neusynthetisierten Rezeptoren zu unterscheiden. Mit Hilfe dieser Methode war es möglich zu zeigen, dass der CRF1R ein recycelnder Rezeptor ist. N2 - Upon ligand binding and receptor activation, G protein-coupled receptors (GPCR) are rapidly desensitized, internalized and subsequently degraded in lysosomes or recycled back to the plasma membrane. Resensitization of the cell is enabled by both recycling receptors and newly synthesized receptors. The overlap of recycling and synthesis processes largely complicates the study of GPCR recycling mechanisms. One aim of this thesis was to develop a new microscopic technique for real-time visualization of GPCR recycling using the photoconvertible Kaede protein allowing to differentiate newly synthesized from recycling receptors. As model proteins, the V1aR (recycling receptor), the V2R (degraded receptor) and the CRF1R were used. In the case of the CRF1R, it was unknown whether this receptor recycles to the plasma membrane following agonist-promoted internalization. The study of the CRF1R recycling behaviour was another objective of this work. As the Kaede protein is fused C-terminally to the GPCRs, an influence on the pharmacological and trafficking properties of the receptors must be excluded. The previously published tetramerization of Kaede, for example, might hinder or even prevent its usability. To assess for the applicability of Kaede, fluorescence correlation spectroscopy experiments were performed and it was demonstrated that Kaede fused to membrane proteins cannot form tetramers in contrast to the soluble form. In vitro studies and experiments in cell culture revealed that both the native and the photoconverted Kaede are equally stable. Moreover Kaede-fused GPCR displayed the same pharmacological and trafficking properties as the untagged or CFP-tagged receptors. Only the expression levels of the Kaede fusion proteins were reduced, yet this did not affect the microscopic experiments. In parallel to these experiments, the interacting amino acids of the tetrameric Kaede were substituted according to the previously published crystal structure of the protein. Unfortunately, these mutations induced protein misfolding thereby causing loss of fluorescence functions. However, since it could be shown that membrane protein-fused Kaede cannot tetramerize, the monomerized Kaede was no more essential for the microscopic study of receptor recycling. In the second part of this work, Kaede-fusions were used to study the recycling behaviour of the CRF1R and the V1aR and V2R control proteins by the novel real-time recycling assay at the laser scanning microscope. To this end, HEK 293 cells expressing the Kaede-fused receptors were treated with agonist to induce receptor internalization. Internalized receptors were selectively photoconverted in endosomes using UV-irradiation and the subcellular fate of the new fluorescence signals was studied. In the case of the CRF1R, signals of the photoconverted receptors could be detected in the plasma membrane indicating that the CRF1R belongs to the family of recycling receptors. The control receptors showed the expected results: The V1aR recycled back to the plasma membrane whereas the V2R did not. These results were confirmed with biochemical and flow cytometry measurements. The CRF1R internalizes in a clathrin-dependent way via the adaptor protein AP2, dynamin and β arrestin. Depending on the stability of the resulting receptor-β-arrestin-complex, two classes of receptors can be differentiated. Class A receptors are recycling receptors undergoing a more transient β-arrestin interaction. In contrast, class B receptors stably interact with β-arrestin and are degraded after internalization. In the case of the CRF1R and V1aR, microscopic analyzes demonstrated that β arrestin transiently interacts with the stimulated CRF1R and V1aR indicating again that these receptors are recycling GPCRs (class A receptors). The V2R, in contrast, revealed a stable interaction (class B receptor). Moreover, it was studied whether the CRF1R recycles rapidly or more slowly to the plasma membrane. Rapidly recycling receptors are recruited out of early endosomes whereas slowly recycling receptors pass the trans-golgi-network or recycling endosomes before reaching the cell surface. Rab11 colocalization studies demonstrated that the CRF1R belongs to the family of slowly recycling receptors. In conclusion, a novel microscopic technique was established allowing to study GPCR recycling in real-time and to differentiate recycling and synthesis processes. Moreover, it was shown that the CRF1R belongs to the family of recycling receptors. The Kaede technique seems to be very well suited to study membrane protein trafficking in general. KW - Corticotropin-Releasing Factor Rezeptor Typ 1 KW - Recycling KW - GPCR KW - Kaede KW - Corticotropin-Releasing Factor Receptor Type 1 KW - Recycling KW - GPCR KW - Kaede Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-34902 ER - TY - THES A1 - El-Saadany, Mohamed Abdel Meged Marawan T1 - Protective effect of dietary antioxidants and plant extracts on acute inflammation and hepatotoxicity in vitro T1 - Protektiver Effekt von Pflanzenextrakten und ernährungsrelevanten Antioxidantien auf akute Entzündung und Hepatotoxizität in vitro N2 - Dietary antioxidants are believed to play an important role in the prevention and treatment of a variety of diseases associated with oxidative stress. Although there is a wide range of dietary antioxidants, the bulk of the research to date has been focused on the nutrient antioxidants vitamin C, E, and carotenoids. Certain relatively uncommon antioxidants such as lipoic acid (LA), and phenolic compounds such as (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and (-)-epigallocatechin gallate (EGCG), have not been extensively investigated although they may exert greater antioxidant potency than that of carotenoids and vitamins. Extracts from selected plants and plant byproducts may represent rich sources for one or more of such antioxidants and therefore exhibit higher effects than a single antioxidant due to the synergistic effects produced between such antioxidants. However, in the last decade a number of epidemiological, animal and in vitro studies have suggested a protective and therapeutic potency of these antioxidants in a broad range of diseases such as cancer, diabetes, atherosclerosis, cataract and acute and chronic neurological disorders. Inflammation, the response of the host toward any infection or injury, plays a central role in the development of many chronic diseases. Several evidences demonstrated the rise of different types of cancer from sites of inflammation. This suggests that active oxygen species and some cytokines generated in the inflamed tissues can cause injury to DNA and ultimately lead to carcinogenesis. Diethylnitrosamine (DEN) is one of the most important environmental carcinogens, present in a variety of foods, alcoholic beverages, tobacco smoke and it can be synthesized endogenously. In addition to the liver it can induce carcinogenesis in other organs like kidney, trachea, lung, esophagus, fore stomach, and nasal cavity. Several epidemiological and laboratory studies indicate that nitroso compounds including DEN may induce hyperplasia and chronic inflammation which is closely associated with the development of hepatocellular carcinoma. Despite increasing evidence on the potential of antioxidants in modulating the etiology of chronic diseases, little is known about their role in inflammation and acute phase response (APR). Therefore the aim of the present work was to study the protective effect of water and solvent extracts of eight plant and plant byproducts including green tea, artichoke, spinach, broccoli, onion and eggplant, orange and potato peels as well as eight antioxidants agents including EC, EGC, ECG, EGCG, ascorbic acid (AA), acetylcysteine (NAC), α-LA, and alpha-tocopherol (α-TOC) toward acute inflammation induced by interleukin-6 (IL-6) and hepatotoxicity induced by DEN in vitro. The negative acute phase proteins (APP), transthyretin (TTR) and retinol-binding protein (RBP) were used as inflammatory biomarkers analyzed by ELISA, whereas neutral red assay was used for evaluating the cytotoxicity. All experiments were performed in vitro using human hepatocarcinoma cell line (HepG2). Additionally the antioxidant activity was measured by TEAC and FRAP assays, phenolic content was measured by Folin–Ciocalteu and characterized by HPLC. Moreover, the microheterogeneity of TTR was detected using immunoprecipitation assay combined with SELDI-TOF MS. Results of present study showed that HepG2 cells provide a simple, sensitive in vitro system for studying the regulation of the negative APP, TTR and RBP under free and inflammatory condition. IL-6, a potent proinflammatory cytokine, in a concentration of 25 ng/ml was able to reduce TTR and RBP secretion by approximately 50-60% after 24h of incubation. With exception of broccoli and water extract of onion which showed pro-inflammatory effects in this study, all other plant extracts, at specific concentrations, were able to elevate TTR secretion in normal condition and even under treatment of IL-6 where the effect was quite lower. Green tea followed by artichoke and potato peel exhibited the highest elevation in TTR concentration which reached 1.1 and 2.5 folds of control in presence and absences of IL-6 respectively. In general Plant extracts were ordered according their anti-inflammatory potency as following: in water extracts; green tea > artichoke > potato peel > orange peel > spinach > eggplant peel, where in solvent extracts; green tea > artichoke > potato peel > spinach > eggplant peel > onion > orange peel. The antiinflammatory effect of water extracts of green tea, artichoke and orange peel were significantly higher than their corresponding solvent extracts whereas water extracts of eggplant-, potato peels and spinach showed lower effect than their solvent extracts. On the other hand α-LA followed by EGCG and ECG exhibited the highest elevation in TTR concentration compared to other antioxidants. The relation between the anti-inflammatory potential and antioxidants activity and phenolic content for the investigated substances was generally weak. This may suggest the involvement of other mechanisms than antioxidants properties for the observed effect. TTR secreted by HepG2 cells has a molecular structure quite similar to the purified standard and serum TTR in which all the three main variants are contained including native, S-cystinylated and Sglutathionylated TTR. Interestingly, a variant with molecular mass of 13453.8 + 8.3 Da has been detected only in TTR secreted by HepG2. Among all investigated antioxidants and plant extracts, six substances were able to elevate the native preferable TTR variant. The potency of these substances can be ordered as following α-LA > NAC > onion > AA > EGCG > green tea. A weak correlation between elevation on TTR and shifting to the native form was observed. Similar weak correlation has also been observed between antioxidants activity and elevation in native TTR. Although DEN was able to induce cell death in a concentration dependent manner, it requires considerably higher concentrations for its effects especially after 24h. This may be attributed to a lack in cytochrome P450 enzymes produced by HepG2. At selected concentrations some antioxidants and plant extracts significantly attenuate DEN cytotoxicity as following: spinach > α-LA > artichoke > orange peel > eggplant peel > α-TOC > onion > AA. Contrary all other substances especially green tea, broccoli, potato peel, and ECG stimulate DEN toxicity. In conclusion, this study demonstrated that selected antioxidants and plant extracts may attenuate the inflammatory process, not only by their antioxidants potency but also by other mechanisms which remain unclear. They may also play a vital role on stabilizing the tetramic structure of TTR and thereby prevent amyloidosis diseases. Lipoic acid represents in this study unique function against inflammation and hepatotoxicity. Despite the protective effect demonstrated by investigated substances, attention should also be given to the pro-oxidant and potential cytotoxic effects produced at higher concentrations. N2 - Substanzen und Lebensmittelinhaltstoffe mit antioxidativer Wirkung spielen eine entscheidende Rolle in Prävention und Behandlung zahlreicher Erkrankungen, die mit oxidativen Stress assoziiert sind. Dabei stehen v. a. die Lebensmittelinhaltsstoffen Vitamin C (Ascorbinsäure, AA), Vitamin E und die Carotinoide im Zentrum der Forschung. Da einige bislang relativ ungebräuchliche Antioxidantien wie Liponsäure (LA) und phenolische Substanzen wie (-)-Epicatechin (EC), (-)-Epigallocatechin(EGC), (-)-Epicatechingallat (ECG), und (-)-Epigallocatechingallat (EGCG) ein größeres antioxidatives Potential als Carotinoide und die Vitamine C und E aufweisen, geraten diese in zunehmendem Maße in den Fokus der Forschung und wecken auch immer mehr das Interesse gesundheitsbewusster Verbraucher. Einige ausgewählte Pflanzenextrakte und Extrake pflanzlicher Nebenprodukte stellen ergiebige Quellen der oben erwähnten Substanzen dar und zeichnen sich daher durch eine höhere Wirksamkeit aus, die teilweise auch auf synergetische Effekte zwischen diesen Antioxidantien zurückzuführen ist. Eine Vielzahl epidemiologischer Studien sowie zahlreiche Tier- und in-vitro-Experimente deuten daher darauf hin, daß die oben erwähnten Antioxidantien bei einer Vielzahl von Erkrankugen, wie Krebs, Diabetes, Arteriosklerose, Katarakt, akute bzw. chronische neurologische Störungen, ein schützendes und therapeutisches Potential entfalten. Entzündungen, als Antwort eines Individuums auf Infektion oder Verletzungen, spielen eine zentrale Rolle bei der Entwicklung vieler chronischer Erkrankungen. So konnten mehrere Studien den Zusammenhang zwischen der Entstehung verschiedener Krebsarten und zugrundeliegender Infektionen belegen. Dies deutet darauf hin, dass reaktive Sauerstoffspezies und einige Zytokinen, die im entzündeten Geweben generiert werden und DNA-Schäden verursachen können, letztendlich auch eine Karzinogenese auslösen können. Diethylnitrosamin (DEN) ist eines der bekanntesten Umweltkarzinogene, daß neben Hepatokarzinomen auch Krebs in Nieren, Trachea, Lunge, Speiseröhre, Magen und Nasenhöhle hervorrufen kann und in vielen Lebensmitteln, alkoholischen Getränke sowie Tabakrauch enthalten ist und darüber hinaus endogen synthetisiert wird. Dabei geht man auf Grundlage mehrere epidemiologischer und Forschungsstudien davon aus, dass durch Nitroso-Verbindungen, u.a. auch DEN, induzierte Hyperplasien und chronische Entzündungen die Entwicklung hepatozellulärer Karzinome begünstigt. Trotz zunehmender Beweise bezüglich des Potentials von Antioxidantien die Ätiologie chronischer Erkrankungen zu modulieren, ist bislang nur sehr wenig über ihre Rolle im Entzündungsprozess und der Akutphasereaktion (APR) bekannt. Deshalb war das Ziel der vorliegenden Arbeit die schützende Wirkung von Extrakten verschiedener Pflanzen und Pflanzennebenprodukten sowie isolierten Antikoxidantien bei akuten Entzündungssituationen zu testen. Dazu wurden wässrige und Lösungsmittelextratke aus acht Pflanzen bzw. deren Nebenprodukten (Grüntee, Artischocke, Spinat, Brokkoli, Zwiebel, Aubergine-, Orangen- und Kartoffelschalen) hergestellt und ihre Wirkung sowie die acht weiterer reiner Antioxidantien (EC, EGC, ECG, EGCG, Ascorbinsäure (AA), Acetylcystein (NAC), LA, und Tocopherol (TOC) in in-vitro-Modellen der akuten Entzündung, induziert durch interleukin-6 (IL-6), bzw. der Hepatoxizität, induziert durch DEN, getestet.. Transthyretin (TTR) und Retinol-Bindungsprotein (RBP), zwei negative Akutphasenproteine (APP) wurden als Entzündungsbiomarker (Analyse per ELISA) und Neutral-Red-Assay als ein Maß für die Cytotoxizität herangezogen. Alle Experimente wurden in-vitro in einer immortalisierten humanen Hepatokarzinom-Zelllinie (HepG2) durchgeführt. Die antioxidativen Kapazität wurde mittels TEAC und FRAP-Methoden evaluiert und der Gesamtphenolgehalt durch die Folin–Ciocalteu-Methode erfasst, wobei die qualitative Charakterisierung über die HPLC erfolgte. Die Mikroheterogenität des TTR wurde durch Immunopräzipitation in Kombination mit SELDI-TOF-MS Technik analysiert. Die Ergebnisse dieser Studie zeigen, dass HepG2-Zellen ein einfaches und empfindliches in-vitro System zur Regulierung von negativen Akutphasenproteinen, TTR und RBP, unter physiologischen und infllammatorischen Bedingungen darstellen. IL-6, ein potentes Pro-Entzündungszytokine, war bei einer 24stündigen Inkubation mit einer Konzentration von 25 ng/ ml in der Lage die Sekretion von TTR und RBP um ca. 50-60% zu reduzieren. Mit Ausnahme von Broccoli und Wasser Extrakt der Zwiebel, die zeigten, proinflammatorischen Effekt Wirkungen in dieser Studie, die alle anderen Pflanzenextrakten, in bestimmten Konzentrationen, waren in der Lage zu erheben TTR Sekretion im normalen, aber auch bei der Behandlung von IL-6 bei denen die Wirkung war niedriger. Grüntee, gefolgt von Artischocken und Kartoffelschälen zeigte die höchste Erhebung in der TTRKonzentration, die erreicht, 1,1 und 2,5 Falten der Kontrolle in Behandlung und ohne Behandlung von IL-6 bzw. Die wässrigen Pflanzenextrakte lassen sich in der folgenden Reihenfolge des anti-Entzündungspotentials einordnen: Grüntee > Artischocke > Kartoffelschalen > Orangenschalen > Spinat > Aubergineschalen, wogegen bei Lösungsmittelextrakte folgende Reihenfolge ermittelt wurde: Grüntee > Artischocke > Kartoffelnschalen > Spinat > Aubergineschalen > Zwiebel > Orangenschalen. Die schützende Wirkung der wässrigen Extrakte von Grüntee, Artischocke und Orangenschalen war signifikant höher als die der entsprechenden Lösungsmittelextrakte. Wohingegen wässrige Extrakte aus Aubergineschalen, Kartoffelschalen, Spinat und Zwiebel weniger effektiv waren. Auf der anderen Seite, LA gefolgt von EGCG und ECG zeigte die höchste Erhebung in der TTRKonzentration im Vergleich zu anderen Antioxidantien. Somit konnte ein schwacher aber Zusammenhang zwischen antinflammatorischem Potential, antioxidativer Aktivität und Phenolgehalt nachgewiesen werden. Daher ist anzunehmen, dass den beobachteten Effekten anderen Mechanismen zu Grunde liegen. Das durch HepG2-Zellen sezernierte TTR erwies eine molekulare Struktur ähnlich der des verwendeten Standards bzw. des TTR aus humanem Serum auf. Es enthielt alle drei Hauptvarianten, einschließlich der nativen, S-cystinylierten und S-glutathionylierten TTR-Formen. Darüber hinaus wurde nur im in-vitro sezerniertem TTR (TTR aus HepG2-Zellen) eine Variante mit einer molekularen Masse von 13453.8 + 8.3 Da nachgewiesen. Von den untersuchten Substanzen wiesen nur sechs Verbindungen die Fähigkeit auf den Anteil der günstigen nativen TTR-Form zu erhöhen aus. Dabei konnte folgende Wirksamkeitsreihenfolge zugeordnet werden : LA > NAC > Zwiebel > AA > EGCG > Grüntee. Eine schwache Korrelation zwischen der Erhöhung der TTRKonzentration und der Verschiebung zu der nativen Form hin wurde festgestellt. Ein ähnlicher Zusammenhang zwischen der antioxidativen Aktivität und dieser Erhöhung wurde auch beobachtet. Obwohl DEN in der Lage war konzentrationsabhängig den Zelltod zu induzieren, war eine wesentlich höhere Konzentration notwendig, um die volle Wirksamkeit während 24stündiger Inkubation zu gewährleisten. Dies mag auf die mangelnde Ausstattung mit Cytochrom-P450-Enzymen, die in den HepG2 Zellen produziert werden, zurück zu führen sein. Ausgewählte Konzentrationen einiger eingesetzter Substanzen führten zu einer signifikanten Schwächung der DEN-induzierten Zytotoxizität mit folgender Wirksamkeit: Spinat > LA > Artischocke > Orangen- > Aubergineschalen > TOC > Zwiebel > AA. Im Gegensatz dazu, stimulierten alle anderen Substanzen, insbesondere Grüntee, Brokkoli, Kartoffelschalen und ECG, die DEN –induzierten Toxizität. Diese Arbeit zeigt somit, dass ausgewählte Antioxidantien und Pflanzenextrakten in der Lage sind, den antinflammatorischen Prozess sowohl durch ihre antioxidative Wirkung als auch durch bislang nicht aufgeklärten Mechanismen grundlegend zu beeinflussen. Sie könnten daher eine entscheidende Rolle bei der Stabilisierung von Proteinstrukturen übernehmen (gezeigt am Beispiel vom TTR) und in diesem Zusammenhang möglicherweise auch zur Prävention von Krankheiten wie Amyloidosen beitragen. Liponsäure überzeugte in dieser Arbeit durch seine einzigartigen Funktion gegenüber Entzündungssituationen und Hepatoxizität. Wie oft beobachtet und durch diese Studie bestätigt, weisen die verwendeten Subsatzen neben der schützenden anti- auch pro-oxidativen Wirkungen auf, wodurch die Notwendigkeit weiterer Untersuchungen zur Erfassung der Zytotoxizität beim Einsatz höherer Konzentration verdeutlicht wird. KW - HepG2 KW - diätetische Antioxidantien KW - Phenole KW - akute Entzündung KW - Hepatotoxizität KW - HepG2 KW - dietary antioxidants KW - phenols KW - acute inflammation KW - hepatotoxicity Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-31585 ER - TY - THES A1 - Küster, Katrin T1 - Die funktionelle Bedeutung des Coxsackie- und Adenovirus Rezeptors (CAR) im kolorektalen Karzinom T1 - Functional role of the Coxsackie and Adenovirus Receptor (CAR) in colorectal carcinomas N2 - Der Coxsackie- und Adenovirus Rezeptor (CAR) ist als Bestandteil von Tight Junctions (TJ) an interzellulären Adhäsionsprozessen beteiligt und scheint eine wichtige Rolle in der Karzinogenese zu spielen. Diese ist jedoch insbesondere bei Entstehung von Darmkrebs weitgehend unklar. Ziel der Arbeit war es daher, die funktionelle Bedeutung, mögliche Interaktionspartner sowie die Expressionsregulation von CAR im kolorektalen Karzinom zu analysieren. In den Zelllinien CaCo2, Colo205, DLD1, HCT116, HT29, SW480 und T84 konnte die Expression von CAR (mRNA und Protein) nachgewiesen werden. Nach stabiler CAR-Überexpression durch Transfektion von CARcDNA in DLD1, HCT116 und SW480 wurde das Zellwachstum gehemmt und eine Abnahme von Migration und Invasion induziert. Eine stabile CAR-Inhibition nach Transfektion von CARsiRNA führte in diesen Zelllinien zum Anstieg der Proliferation sowie zu verstärkter Migrations- und Invasionsaktivität, die in DLD1 mit morphologischen Änderungen einhergingen. Eine Genexpressionsanalyse der Zelllinie DLD1 mit CAR-Inhibition identifizierte α-Catenin als das am stärksten regulierte Gen. Obwohl keine direkte Interaktion beider Proteine detektiert werden konnte, führte eine stabile Re-Expression von α-Catenin in DLD1 mit stabiler CAR-Inhibition zu einer deutlichen Reduktion von Proliferation, Migration und Invasion sowie zu einem Rückgang der zellmorphologischen Änderungen. Um den Einfluss von Differenzierung auf die Regulation der CAR-Expression zu untersuchen, erfolgte eine Behandlung aller Zelllinien mit Natriumbutyrat. Dies führte in fünf der sieben Zelllinien zu einer Aktivierung des CAR-Promotors sowie zu einer gesteigerten Expression und Immunoreaktivität von CAR an der Zelloberfläche. Die Zelllinie CaCo2 zeigte nach spontaner Differenzierung durch 21-tägiges Wachstum post Konfluenz ebenfalls eine verstärkte CAR-mRNA-Expression sowie eine erhöhte CAR-Präsenz an der Zelloberfläche. Die gewonnenen Daten konnten die funktionelle Bedeutung von CAR für die Kolonkarzinogenese sowie den Einfluss von α-Catenin auf diese Funktion deutlich machen. Es wurde gezeigt, dass die Expressionsregulation sowie die subzelluläre Verteilung von CAR durch den zellulären Differenzierungsstatus beeinflusst werden kann. N2 - The Coxsackie and Adenovirus Receptor (CAR) is a transmembrane compound of the tight junctions in polarized epithelial cells mediating cellular adhesion. CAR was suggested to play a functional role in the development of epithelial malignomas but detailed knowledge is still lacking, especially for the colorectal carcinoma. Therefore, the functional impact and regulation of CAR expression in human colorectal carcinoma cell models were investigated. CAR protein and mRNA was detectable in the cell lines CaCo2, Colo205, DLD1, HCT116, HT29, SW480 and T84. Stable CAR over expression by transfection of CARcDNA in DLD1, HCT116 and SW480 led to reduced proliferation in vitro and in vivo. Also reduced migration and invasion were observed. Stable CAR inhibition by transfection of CARsiRNA in the same cell lines resulted in increased migration and invasion. In DLD1 morphological changes were found after CAR inhibition. Differential gene expression was detected in DLD1 cells with stable CAR inhibition revealing an 18-fold decrease in α-Catenin gene expression. Loss of α-Catenin was obtained on protein level, too. Although no direct interaction between CAR and α-Catenin could be proven ectopic re-expression of α-Catenin in DLD1 with CAR inhibition reversed the determined functional and morphological effects of a CAR knock down. Then, the impact of differentiation on regulation of CAR expression was investigated. Sodium butyrate treatment induced differentiation in all cell lines (determined by alkaline phosphatase activity), which was paralleled by an increase of CAR immunoreactivity at the plasma membrane in all cell lines but CaCo2. However, CAR protein and mRNA expression, as well as CAR gene promoter activity increased in 5 cell lines only, whereas in SW480 and CaCo2 a down regulation was observed. Spontaneous differentiation of CaCo2 after a growth period of 21 days post confluence resulted in up regulation of CAR mRNA expression as well as increased CAR presence at the plasma membrane. The data suggest that CAR plays a crucial role in the carcinogenesis of colorectal carcinoma which could be influenced by α-Catenin interaction. Differentiation determines the regulation of CAR expression and the subcellular distribution of CAR in colon cancer cells. KW - Coxsackie- und Adenovirus Rezeptor KW - kolorektales Karzinom KW - Tight Junctions KW - alpha-Catenin KW - Differenzierung KW - Coxsackie and Adenovirus Receptor KW - colorectal carcinoma KW - tight junctions KW - alpha-Catenin KW - differentiation Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-31617 ER - TY - THES A1 - Frey, Simone K. T1 - Investigations on extra- and intracellular retinol-binding proteins T1 - Untersuchungen zu extra- und intrazellulären Retinol-Bindungsproteinen N2 - The fat-soluble vitamin A, which is chemically referred to retinol (ROH), is known to be essential for the process of vision, the immune system but also for cell differentiation and proliferation. Recently, ROH itself has been reported to be involved in adipogenesis and a ROH transport protein, the retinol-binding protein 4 (RBP4), in insulin resistance and type 2 diabetes. However, there is still considerable scientific debate about this relation. With the increasing amount of studies investigating the relation of ROH in obesity and type 2 diabetes, basic research is an essential prerequisite for interpreting these results. This thesis enhances the knowledge on this relation by reviewing ROH metabolism on extra- and intracellular level. Aim 1: In the blood stream ROH is transported in a complex with RBP4 and a second protein, transthyretin (TTR), to the target cells. The levels of RBP4 and TTR are influenced by several factors but mainly by liver and kidney function. The reason for that is that liver and the kidneys are the sites of RBP4 synthesis and catabolism, respectively. Interestingly, obesity and type 2 diabetes involve disorders of the liver and the kidneys. Therefore the aim was to investigate factors that influence RBP4 and TTR levels in relation to obesity and type 2 diabetes (Part 1). Aim 2: Once arrived in the target cell ROH is bound to cellular retinol-binding protein type I (CRBP-I) and metabolised: ROH can either be stored as retinylesters or it can be oxidised to retinoic acid (RA). By acting as a transcription factor in the nucleus RA may influence processes such as adipogenesis. Therefore vitamin A has been postulated to be involved in obesity and type 2 diabetes. CRBP-I is known to mediate the storage of ROH in the liver, but the extra-hepatic metabolism and the functions of CRBP-I are not well known. This has been investigated in Part 2 of this work. Material & Methods: RBP4 and TTR levels were investigated by ELISA in serum samples of human subjects with overweight, type 2 diabetes, kidney or liver dysfunction. Molecular alterations of the RBP4 and TTR protein structure were analysed by MALDI-TOF mass spectrometry. The functions of intracellular CRBP-I were investigated in CRBP-I knock-out mice in liver and extra-hepatic tissues by measuring ROH levels as well as the levels of its storage form, the retinylesters, using reverse phase HPLC. The postprandial uptake of ROH into tissues was analysed using labelled ROH. The mRNA levels of enzymes that metabolize ROH were examined by real-time polymerase chain reaction (RCR). Results: The previous published results showing increased RBP4 levels in type 2 diabetic patients could not be confirmed in this work. However, it could be shown that during kidney dysfunction RBP4 levels are increased and that RBP4 and TTR levels are decreased during liver dysfunction. The important new finding of this work is that increased RBP4 levels in type 2 diabetic mice were increased when kidney function was decreased. Thus an increase in RBP4 levels in type 2 diabetes may be the effect of a reduced kidney function which is common in type 2 diabetes. Interestingly, during severe kidney dysfunction the molecular structure of RBP4 and TTR was altered in a specific manner which was not the case during liver diseases and type 2 diabetes. This underlines the important function of the kidneys in RBP4 metabolism. CRBP-I has been confirmed to be responsible for the ROH storage in the liver since CRBP-I knock-out mice had decreased ROH and retinylesters (the storage form of ROH) levels in the liver. Interestingly, in the adipose tissue (the second largest ROH storage tissue in the body) ROH and retinylesters levels were higher in the CRBP-I knock-out compared to the wild-type mice. It could be shown in this work that a different ROH binding protein, cellular retinol-binding protein type III, is upregulated in CRBP-I knock-out mice. Moreover enzymes were identified which mediate very efficiently ROH esterification in the adipose tissue of the knock-out mice. In the pancreas there was a higher postprandial ROH uptake in the CRBP-I knock-out compard to wild-type mice. Even under a vitamin A deficient diet the knock-out animals had ROH and retinylesters levels which were comparable to wild-type animals. These results underline the important role of ROH for insulin secretion in the pancreas. Summing up, there is evidence that RBP4 levels are more determined by kidney function than by type 2 diabetes and that specific molecular modifications occur during kidney dysfunction. The results in adipose tissue and pancreas of CRBP-I knock-out mice support the hypothesis that ROH plays an important role in glucose and lipid metabolism. N2 - Vitamin A gehört zur Gruppe der fettlöslichen Vitamine und wird chemisch als Retinol bezeichnet. Es ist essentiell für den Prozess des Sehvorgangs und der Zelldifferenzierung und kann daher bestimmte Entwicklungsprozesse wie die Bildung des Fettgewebes beeinflussen. Aufgrund seiner Fettlöslichkeit muss Retinol im Blut (= extrazellulär) sowie in der Zelle (= intrazellulär) an sogenannte Transport-Moleküle, die Retinol-bindenden Proteine (RBPs) gebunden werden. Die zwei bekanntesten Vertreter der RBPs sind das Retinol-bindende Protein 4 (RBP4) und das intrazelluläre Retinol-bindende Protein Typ I (CRBP-I). RBP4 transportiert Vitamin A im Blut von der Leber zur Zielzelle und zum Abbauorgan für Vitamin A, der Niere. CRBP-I ist in der Leber für die Speicherung von Vitamin A zuständig. In den letzten Jahren wurden neben der Beteiligung des Retinols an der Bildung des Fettgewebes auch Studien veröffentlicht, in denen ein Zusammenhang zwischen erhöhten RBP4-Werte im Blut und Typ-2-Diabetes gezeigt wurde. Bis heute ist der mögliche Zusammenhang zwischen RBP4, CRBP-I und Übergewicht nicht ausreichend erforscht. Im ersten Teil der Arbeit war daher das Ziel, Einflussfaktoren, die zu Veränderungen der RBP4-Werte im Blut führen können, zu untersuchen. Dazu wurden Blutproben von Personen mit Übergewicht und/oder Typ-2-Diabetes und Patienten mit Nierenfunktionsstörungen oder mit Leberfunktionsstörungen analysiert. Es konnte gezeigt werden, dass bereits geringe Nierenfunktionsstörungen zu erhöhten RBP4-Konzentrationen im Blut führten. Bei Typ-2-Diabetikern, die sehr oft an Nierenfunktionsstörungen leiden, war eine Erhöhung der RBP4-Konzentration mit einer Abnahme der Nierenfunktion verbunden. Somit lässt sich zusammenfassen, dass nicht Typ-2-Diabetes sondern vielmehr die dabei auftretenden Nierenfunktionsstörungen zu einer Erhöhung der RBP4-Werte führen. Bei Lebererkrankten konnte ein Absinken der RBP4-Werte nachgewiesen werden, was der verminderten Bildung von RBP4 in der Leber bei diesen Patienten zuzuschreiben ist. Im zweiten Teil sollte der Frage nachgegangen werden, wie Retinol intrazellulär verstoffwechselt wird. Dabei lag der Fokus auf der Erforschung der bisher nicht bekannten Funktionen von CRBP-I im Fettgewebe und der Bauchspeicheldrüse. Zur Untersuchung der Funktionen von CRBP-I wurden Mäuse gezüchtet, bei denen das Gen für CRBP-I gelöscht wurde. Da CRBP-I für die Speicherung von Vitamin A in der Leber verantwortlich ist, zeigen diese Mäuse sehr geringe Vitamin-A-Speicher in der Leber. Das gleiche zeigte sich für die Bauchspeicheldrüse, die für die Sekretion von Insulin Vitamin A benötigt: In den Mäusen ohne CRBP-I waren die Retinol-Werte drastisch gesunken. Interessanterweise zeigte sich im Fettgewebe ein gegenteiliges Bild: Die Konzentrationen an Retinol und dessen Speicher waren in den Mäusen ohne CRBP-I höher im Vergleich zu den normalen Mäusen. Mit bestimmten Nachweismethoden konnte herausgefunden werden, dass Retinol im Fettgewebe an ein anderes RBP, das CRBP-III, gebunden wird und dadurch effektiver gespeichert werden kann als durch CRBP-I. KW - Vitamin A KW - retinol KW - RBP KW - Retinol-Bindungsprotein 4 KW - Diabetes KW - Vitamin A KW - retinol KW - RBP KW - Retinol-binding protein 4 KW - diabetes Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-31428 ER -