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Institute
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Die Rolle von Glucosetransportern der GLUT-Familie für die Glucosehomöostase und als Glucosesensor
(2007)
Untersuchung des Recyclings Kaede-fusionierter Corticotropin-Releasing-Factor Rezeptoren Typ 1
(2009)
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.
Botulinum neurotoxin (BoNT) is produced by the anaerobic bacterium Clostridium botulinum. It is one of the most potent toxins found in nature and can enter motor neurons (MN) to cleave proteins necessary for neurotransmission, resulting in flaccid paralysis. The toxin has applications in both traditional and esthetic medicine. Since BoNT activity varies between batches despite identical protein concentrations, the activity of each lot must be assessed. The gold standard method is the mouse lethality assay, in which mice are injected with a BoNT dilution series to determine the dose at which half of the animals suffer death from peripheral asphyxia. Ethical concerns surrounding the use of animals in toxicity testing necessitate the creation of alternative model systems to measure the potency of BoNT.
Prerequisites of a successful model are that it is human specific; it monitors the complete toxic pathway of BoNT; and it is highly sensitive, at least in the range of the mouse lethality assay. One model system was developed by our group, in which human SIMA neuroblastoma cells were genetically modified to express a reporter protein (GLuc), which is packaged into neurosecretory vesicles, and which, upon cellular depolarization, can be released – or inhibited by BoNT – simultaneously with neurotransmitters. This assay has great potential, but includes the inherent disadvantages that the GLuc sequence was randomly inserted into the genome and the tumor cells only have limited sensitivity and specificity to BoNT. This project aims to improve these deficits, whereby induced pluripotent stem cells (iPSCs) were genetically modified by the CRISPR/Cas9 method to insert the GLuc sequence into the AAVS1 genomic safe harbor locus, precluding genetic disruption through non-specific integrations. Furthermore, GLuc was modified to associate with signal peptides that direct to the lumen of both large dense core vesicles (LDCV), which transport neuropeptides, and synaptic vesicles (SV), which package neurotransmitters. Finally, the modified iPSCs were differentiated into motor neurons (MNs), the true physiological target of BoNT, and hypothetically the most sensitive and specific cells available for the MoN-Light BoNT assay.
iPSCs were transfected to incorporate one of three constructs to direct GLuc into LDCVs, one construct to direct GLuc into SVs, and one “no tag” GLuc control construct. The LDCV constructs fused GLuc with the signal peptides for proopiomelanocortin (hPOMC-GLuc), chromogranin-A (CgA-GLuc), and secretogranin II (SgII-GLuc), which are all proteins found in the LDCV lumen. The SV construct comprises a VAMP2-GLuc fusion sequence, exploiting the SV membrane-associated protein synaptobrevin (VAMP2). The no tag GLuc expresses GLuc non-specifically throughout the cell and was created to compare the localization of vesicle-directed GLuc.
The clones were characterized to ensure that the GLuc sequence was only incorporated into the AAVS1 safe harbor locus and that the signal peptides directed GLuc to the correct vesicles. The accurate insertion of GLuc was confirmed by PCR with primers flanking the AAVS1 safe harbor locus, capable of simultaneously amplifying wildtype and modified alleles. The PCR amplicons, along with an insert-specific amplicon from candidate clones were Sanger sequenced to confirm the correct genomic region and sequence of the inserted DNA. Off-target integrations were analyzed with the newly developed dc-qcnPCR method, whereby the insert DNA was quantified by qPCR against autosomal and sex-chromosome encoded genes. While the majority of clones had off-target inserts, at least one on-target clone was identified for each construct.
Finally, immunofluorescence was utilized to localize GLuc in the selected clones. In iPSCs, the vesicle-directed GLuc should travel through the Golgi apparatus along the neurosecretory pathway, while the no tag GLuc should not follow this pathway. Initial analyses excluded the CgA-GLuc and SgII-GLuc clones due to poor quality protein visualization. The colocalization of GLuc with the Golgi was analyzed by confocal microscopy and quantified. GLuc was strongly colocalized with the Golgi in the hPOMC-GLuc clone (r = 0.85±0.09), moderately in the VAMP2-GLuc clone (r = 0.65±0.01), and, as expected, only weakly in the no tag GLuc clone (r = 0.44±0.10). Confocal microscopy of differentiated MNs was used to analyze the colocalization of GLuc with proteins associated with LDCVs and SVs, SgII in the hPOMC-GLuc clone (r = 0.85±0.08) and synaptophysin in the VAMP2-GLuc clone (r = 0.65±0.07). GLuc was also expressed in the same cells as the MN-associated protein, Islet1.
A significant portion of GLuc was found in the correct cell type and compartment. However, in the MoN-Light BoNT assay, the hPOMC-GLuc clone could not be provoked to reliably release GLuc upon cellular depolarization. The depolarization protocol for hPOMC-GLuc must be further optimized to produce reliable and specific release of GLuc upon exposure to a stimulus. On the other hand, the VAMP2-GLuc clone could be provoked to release GLuc upon exposure to the muscarinic and nicotinic agonist carbachol. Furthermore, upon simultaneous exposure to the calcium chelator EGTA, the carbachol-provoked release of GLuc could be significantly repressed, indicating the detection of GLuc was likely associated with vesicular fusion at the presynaptic terminal. The application of the VAMP2-GLuc clone in the MoN-Light BoNT assay must still be verified, but the results thus far indicate that this clone could be appropriate for the application of BoNT toxicity assessment.
Die Phyllosphäre
(2015)
The prevalence of depression and anxiety is increased in obese patients compared to healthy humans, which is partially due to a shared pathogenesis, including insulin resistance and inflammation. These factors are also linked to intestinal dysbiosis. Additionally, the chronic consumption of diets rich in saturated fats results in body weight gain, hormonal resistances and unfavorable changes in the microbiome composition. The intake of Lactobacilli has already been shown to improve dysbiosis along with metabolism and mood. Yet, the beneficial role and the underlying mechanism of Lactobacillus rhamnosus GG (LGG) to improve emotional behavior in established diet-induced obese conditions are, so far, unknown. To characterize the role of LGG in diet-induced obesity, female and male C57BL/6N mice were fed a semi-synthetic low-fat diet (LFD, 10 % kcal from fat) or a conventional high-fat diet (HFD, 45 % kcal from fat) for initial 6 weeks, which was followed by daily oral gavage of vehicle or 1x10^8 CFU of LGG until the end of the experiment. Mice were subjected to basic metabolic and extensive behavioral phenotyping, with a focus on emotional behavior. Moreover, composition of cecal gut microbiome, metabolomic profile in plasma and cerebrospinal fluid was investigated and followed by molecular analyses. Both HFD-feeding and LGG application resulted in sex-specific differences. While LGG prevented the increase of plasma insulin, adrenal gland weight and hyperactivity in diet-induced obese female mice, there was no regulation of anxiodepressive-like behavior. In contrast, metabolism of male mice did not benefit from LGG application, but strikingly, LGG decreased specifically depressive-like behavior in the Mousetail Suspension Test which was confirmed by the Splash Test characterizing motivation for ’self-care’. The microbiome analysis in male mice revealed that HFD-feeding, but not LGG application, altered cecal microbiome composition, indicating a direct effect of LGG on behavioral regulation. However, in female mice, both HFD-feeding and LGG application resulted in changes of microbiome composition, which presumably affected metabolism. Moreover, as diet-induced obese female mice unexpectedly did not exhibit anxiodepressive-like behavior, follow-up analyses were conducted in male mice. Here, HFD-feeding significantly altered abundance of plasma lipids whereas LGG decreased branched chain amino acids which associated with improved emotional behavior. In nucleus accumbens (NAcc) and VTA/SN, which belong to the dopaminergic system, LGG restored HFD-induced decrease of tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, on gene expression level. Lastly, transcriptome analysis in the NAcc identified gene expression of cholecystokinin as a potential mediator of the effect of LGG on HFD-induced emotional alterations. In summary, this thesis revealed the beneficial effects of LGG application on emotional alterations in established diet-induced obesity. Furthermore, both HFD-feeding and LGG treatment exhibited sex-specific effects, resulting in metabolic improvements in female mice while LGG application mitigated depressive-like behavior in obese male mice along with a molecular signature of restored dopamine synthesis and neuropeptide signaling.
Darmkrebs ist die zweithäufigste malignombedingte Todesursache in den westlichen Industrieländern. Durch eine frühzeitige Diagnose besteht jedoch eine hohe Chance auf Heilung. Der Goldstandard zur Darmkrebsfrüherkennung ist gegenwärtig die Koloskopie. Eine Darmspiegelung ist jedoch invasiv und mit Unannehmlichkeiten für den Patienten verbunden. Die Akzeptanz in der Bevölkerung ist daher gering. Ziel des BMBF- Projektes „Entwicklung eines nichtinvasiven Nachweissystems zur Früherkennung von humanem Darmkrebs“, in dessen Rahmen diese Arbeit entstand, ist die Bereitstellung eines nichtinvasiven Nachweisverfahrens zur Darmkrebsfrüherkennung. Der Nachweis soll über die Detektion von aus neoplastischen Zellen stammender DNA in Stuhl erfolgen. Die Entartung dieser Zellen beruht auf Veränderungen im Erbgut, welches unter anderem Mutationen sind. Im ersten Teil des BMBF-Projektes wurde ein Set von Mutationen zusammengestellt, welches eine hohe Sensitivität für Vorstufen von Darmkrebs aufweist. Ziel dieser Arbeit war es, eine Nachweismethode für die zuvor identifizierten Punktmutationen zu entwickeln. Das Nachweisverfahren musste dabei unempfindlich gegen einen hohen Hintergrund nichtmutierter DNA sein, da im Stuhl geringe Mengen DNA aus neoplastischen Zellen bei einem hohen Hintergrund von DNA aus gesunden Zellen vorliegen. Hierzu wurden Plasmidmodellsysteme für die aus dem Marker-Set stammenden Genfragmente BRAF und dessen Mutante V600E, CTNNB1 und T41I, T41A, S45P und K-ras G12C hergestellt. Mit Hilfe dieser Plasmidmodellsysteme wurde dann das Nachweissystem entwickelt. Der entscheidende Schritt für die Detektion von Punktmutationen bei hohem Wildtypüberschuss ist eine vorhergehende Anreicherung. In der vorliegenden Arbeit wurde dazu die Methode der LNA-clamp-PCR (locked nucleic acid) etabliert. Die Bewertung der erzielten Anreicherung erfolgte über das relative Detektionslimit. Zur Bestimmung des Detektionslimits wurde die Schmelzkurvenanalyse von Hybridisierungssonden eingesetzt; diese wurde im Rahmen dieser Arbeit für die drei oben genannten Genfragmente und ihre Mutanten entwickelt. Die LNA-clamp-PCR wird in Anwesenheit eines LNA-Blockers durchgeführt. Das Nukleotidanalogon LNA weist im Vergleich zu DNA eine erhöhte Affinität zu komplementären DNA-Strängen auf. Gleichzeitig kommt es bei Anwesenheit einer Basenfehlpaarung zu einer größeren Destabilisierung der Bindung. Als Blocker werden kurze LNA-DNA-Hybridoligonukleotide eingesetzt, die den mutierten Sequenzbereich überspannen und selbst der Wildtypsequenz entsprechen. Durch Bindung an die Wildtypsequenz wird deren Amplifikation während der PCR verhindert (clamp = arretieren, festklemmen). Der Blocker selbst wird dabei nicht verlängert. Der Blocker bindet unter optimalen Bedingungen jedoch nicht an die mutierte Sequenz. Die Mutante wird daher ungehindert amplifiziert und somit gegenüber dem Wildtyp-Fragment angereichert. Die Position des Blockers kann im Bindungsbereich eines der Primer sein und hier dessen Hybridisierung an dem Wildtyp-Fragment verhindern oder zwischen den beiden Primern liegen und so die Synthese durch die Polymerase inhibieren. Die Anwendbarkeit beider Systeme wurde in dieser Arbeit gezeigt. Die LNA-clamp-PCR mit Primerblocker wurde für BRAF etabliert. Es wurde ein Detektionslimit von mindestens 1:100 erzielt. Die LNA-clamp-PCR mit Amplifikationsblocker wurde erfolgreich für BRAF, K-ras und CTNNB1: T41I, T41A mit einem Detektionslimit von 1:1000 bis 1:10 000 entwickelt. In Stuhlproben liegt DNA aus neoplastischen Zellen nach Literaturangaben zu einem Anteil von 1% bis 0,1% vor. Die LNA-clamp-PCR weist also mit Amplifikationsblockern ein ausreichend hohes Detektionslimit für die Analyse von Stuhlproben auf. Durch die erfolgreiche Etablierung der Methode auf drei verschiedenen Genfragmenten und vier unterschiedlichen Punktmutationen konnte deren universelle Einsetzbarkeit gezeigt werden. Für die Ausweitung der LNA-clamp-PCR auf die übrigen Mutationen des Marker-Sets wurden Richtlinien ausgearbeitet und die Blockereffizienz als Kennzahl eingeführt. Die LNA-clamp-PCR ist ein schnelles, kostengünstiges Verfahren, welches einen geringen Arbeitsaufwand erfordert und wenig fehleranfällig ist. Sie ist somit ein geeignetes Anreicherungsverfahren für Punktmutationen in einem diagnostischen System zur Darmkrebsfrüherkennung. Darüber hinaus kann die LNA-clamp-PCR auch in anderen Bereichen, in denen die Detektion von Punktmutationen in einem hohen Wildtyphintergrund erforderlich ist, eingesetzt werden.
The development of type 2 diabetes (T2D) is driven by genetic as well as life style factors. However, even genetically identical female NZO mice on a high-fat diet show a broad variation in T2D onset. The main objective of this study was to elucidate and investigate early epigenetic determinants of type 2 diabetes. Prior to other experiments, early fat content of the liver (<55.2 HU) in combination with blood glucose concentrations (>8.8 mM) were evaluated as best predictors of diabetes in NZO females. Then, DNA methylome and transcriptome were profiled to identify molecular pathophysiological changes in the liver before diabetes onset. The major finding of this thesis is that alterations in the hepatic DNA methylome precede diabetes onset. Of particular interest were 702 differentially methylated regions (DMRs), of which 506 DMRs had genic localization. These inter-individual DMRs were enriched by fivefold in the KEGG pathway type 2 diabetes mellitus, independent of the level of gene expression, demonstrating an epigenetic predisposition toward diabetes. Interestingly, among the list of hepatic DMRs, eleven DMRs were associated with known imprinted genes in the mouse genome. Thereby, six DMRs (Nap1l5, Mest, Plagl1, Gnas, Grb10 and Slc38a4) localized to imprinting control regions, including five iDMRs that exhibited hypermethylation in livers of diabetes-prone mice. This suggests that gain of DNA methylation in multiple loci of the paternal alleles has unfavourable metabolic consequences for the offspring. Further, the comparative liver transcriptome analysis demonstrated differences in expression levels of 1492 genes related to metabolically relevant pathways, such as citrate cycle and fatty acid metabolism. The integration of hepatic transcriptome and DNA methylome indicated that 449 differentially expressed genes were potentially regulated by DNA methylation, including genes implicated in insulin signaling. In addition, liver transcriptomic profiling of diabetes-resistant and diabetes-prone mice revealed a potential transcriptional dysregulation of 17 hepatokines, in particular Hamp. The hepatic expression of Hamp was decreased by 52% in diabetes-prone mice, on account of an increase in DNA methylation of promoter CpG-118. Hence, HAMP protein levels were lower in mice prone to develop diabetes, which correlated to higher liver triglyceride levels.. In sum, the identified DNA methylation changes appear to collectively favor the initiation and progression of diabetes in female NZO mice. In near future, epigenetic biomarkers are likely to contribute to improved diagnosis for T2D.
The relationship between nutrition and the development of chronic diseases including metabolic syndrome, diabetes mellitus, cancer and cardiovascular disease has been well studied. On the other hand, changes in the GH-IGF-1 axis in association with nutrition-related diseases have been reported. The interplay between GH, total IGF-1 and different inhibitory and stimulatory kinds of IGF-1 binding proteins (IGFBPs) results in IGF-1 bioactivity, the ability of IGF-1 to induce phosphorylation of its receptor and consequently its signaling. Moreover, IGF-1 bioactivity is sufficient to reflect any change in the GH-IGF-1 system. Accumulating evidence suggests that both of high protein diet, characterized by increased glucagon secretion, and insulin-induced hypoglycemia increase mortality rate and the mechanisms are unclear. However both of glucagon and insulin-induced hypoglycemia are potent stimuli of GH secretion. The aim of the current study was to identify the impact of glucagon and insulin-induced hypoglycemia on IGF-1 bioactivity as possible mechanisms. In a double-blind placebo-controlled study, glucagon was intramuscularly administrated in 13 type 1 diabetic patients (6 males /7 females; [BMI]: 24.8 ± 0.95 kg/m2), 11 obese subjects (OP; 5/ 6; 34.4 ± 1.7 kg/m2), and 13 healthy lean participants (LP; 6/ 7; 21.7 ± 0.6 kg/m2), whereas 12 obese subjects (OP; 6/ 6; 34.4 ± 1.7 kg/m2), and 13 healthy lean participants (LP; 6/ 7; 21.7 ± 0.6 kg/m2) performed insulin tolerance test in another double-blind placebo-controlled study and changes in GH, total IGF-1, IGF binding proteins (IGFBPs) and IGF-1 bioactivity, measured by the cell-based KIRA method, were investigated. In addition, the interaction between the metabolic hormones (glucagon and insulin) and the GH-IGF-1 system on the transcriptional level was studied using mouse primary hepatocytes. In this thesis, glucagon decreased IGF-1 bioactivity in humans independently of endogenous insulin levels, most likely through modulation of IGFBP-1 and-2 levels. The glucagon-induced reduction in IGF-1 bioactivity may represent a novel mechanism underlying the impact of glucagon on GH secretion and may explain the negative effect of high protein diet related to increased cardiovascular risk and mortality rate. In addition, insulin-induced hypoglycemia was correlated with a decrease in IGF-1 bioactivity through up-regulation of IGFBP-2. These results may refer to a possible and poorly explored mechanism explaining the strong association between hypoglycemia and increased cardiovascular mortality among diabetic patients.
Diet is a major force influencing the intestinal microbiota. This is obvious from drastic changes in microbiota composition after a dietary alteration. Due to the complexity of the commensal microbiota and the high inter-individual variability, little is known about the bacterial response at the cellular level. The objective of this work was to identify mechanisms that enable gut bacteria to adapt to dietary factors. For this purpose, germ-free mice monoassociated with the commensal Escherichia coli K-12 strain MG1655 were fed three different diets over three weeks: a diet rich in starch, a diet rich in non-digestible lactose and a diet rich in casein. Two dimensional gel electrophoresis and electrospray tandem mass spectrometry were applied to identify differentially expressed proteins of E. coli recovered from small intestine and caecum of mice fed the lactose or casein diets in comparison with those of mice fed the starch diet. Selected differentially expressed bacterial proteins were characterised in vitro for their possible roles in bacterial adaptation to the various diets. Proteins belonging to the oxidative stress regulon oxyR such as alkyl hydroperoxide reductase subunit F (AhpF), DNA protection during starvation protein (Dps) and ferric uptake regulatory protein (Fur), which are required for E. coli’s oxidative stress response, were upregulated in E. coli of mice fed the lactose-rich diet. Reporter gene analysis revealed that not only oxidative stress but also carbohydrate-induced osmotic stress led to the OxyR-dependent expression of ahpCF and dps. Moreover, the growth of E. coli mutants lacking the ahpCF or oxyR genes was impaired in the presence of non-digestible sucrose. This indicates that some OxyR-dependent proteins are crucial for the adaptation of E. coli to osmotic stress conditions. In addition, the function of two so far poorly characterised E. coli proteins was analysed: 2 deoxy-D gluconate 3 dehydrogenase (KduD) was upregulated in intestinal E. coli of mice fed the lactose-rich diet and this enzyme and 5 keto 4 deoxyuronate isomerase (KduI) were downregulated on the casein-rich diet. Reporter gene analysis identified galacturonate and glucuronate as inducers of the kduD and kduI gene expression. Moreover, KduI was shown to facilitate the breakdown of these hexuronates, which are normally degraded by uronate isomerase (UxaC), altronate oxidoreductase (UxaB), altronate dehydratase (UxaA), mannonate oxidoreductase (UxuB) and mannonate dehydratase (UxuA), whose expression was repressed by osmotic stress. The growth of kduID-deficient E. coli on galacturonate or glucuronate was impaired in the presence of osmotic stress, suggesting KduI and KduD to compensate for the function of the regular hexuronate degrading enzymes under such conditions. This indicates a novel function of KduI and KduD in E. coli’s hexuronate metabolism. Promotion of the intracellular formation of hexuronates by lactose connects these in vitro observations with the induction of KduD on the lactose-rich diet. Taken together, this study demonstrates the crucial influence of osmotic stress on the gene expression of E. coli enzymes involved in stress response and metabolic processes. Therefore, the adaptation to diet-induced osmotic stress is a possible key factor for bacterial colonisation of the intestinal environment.
Aging is associated with bone loss, which can lead to osteoporosis and high fracture risk. This coincides with the enhanced formation of bone marrow adipose tissue (BMAT), suggesting a negative effect of bone marrow adipocytes on skeletal health. Increased BMAT formation is also observed in pathologies such as obesity, type 2 diabetes and osteoporosis. However, a subset of bone marrow adipocytes forming the constitutive BMAT (cBMAT), arise early in life in the distal skeleton, contain high levels of unsaturated fatty acids and are thought to provide a physiological function. Regulated BMAT (rBMAT) forms during aging and obesity in proximal regions of the bone and contain a large proportion of saturated fatty acids. Paradoxically, BMAT accumulation is also enhanced during caloric restriction (CR), a life-span extending dietary intervention. This indicates, that different types of BMAT can form in response to opposing nutritional stimuli with potentially different functions.
To this end, two types of nutritional interventions, CR and high fat diet (HFD), that are both described to induce BMAT accumulation were carried out. CR markedly increased BMAT formation in the proximal tibia and led to a higher proportion of unsaturated fatty acids, making it similar to the physiological cBMAT. Additionally, proximal and diaphyseal tibia regions displayed higher adiponectin expression. In aged mice, CR was associated with an improved trabecular bone structure. Taken together, these findings demonstrate, that the type of BMAT that forms during CR might provide beneficial effects for local bone stem/progenitor cells and metabolic health. The HFD intervention performed in this thesis showed no effect on BMAT accumulation and bone microstructure. RNA Seq analysis revealed alterations in the composition of the collagen-containing extracellular matrix (ECM).
In order to investigate the effects of glucose homeostasis on osteogenesis, differentiation capacity of immortalized multipotent mesenchymal stromal cells (MSCs) and osteochondrogenic progenitor cells (OPCs) was analyzed. Insulin improved differentiation in both cell types, however, combination of with a high glucose concentration led to an impaired mineralization of the ECM. In the MSCs, this was accompanied by the formation of adipocytes, indicating negative effects of the adipocytes formed during hyperglycemic conditions on mineralization processes. However, the altered mineralization pattern and structure of the ECM was also observed in OPCs, which did not form any adipocytes, suggesting further negative effects of a hyperglycemic environment on osteogenic differentiation.
In summary, the work provided in this thesis demonstrated that differentiation commitment of bone-resident stem cells can be altered through nutrient availability, specifically glucose. Surprisingly, both high nutrient supply, e.g. the hyperglycemic cell culture conditions, and low nutrient supply, e.g. CR, can induce adipogenic differentiation. However, while CR-induced adipocyte formation was associated with improved trabecular bone structure, adipocyte formation in a hyperglycemic cell-culture environment hampered mineralization. This thesis provides further evidence for the existence of different types of BMAT with specific functions.
Microbiota analyses of patients suffering from various diseases suggest a beneficial role of Akkermansia muciniphila in the maintenance of health, whereas several studies in animal models of intestinal inflammation report that this organism may aggravate inflammation. Therefore, it is important to clarify under which circumstances A. muciniphila exerts negative effects in the intestine of its host.
The previously reported observation that A. muciniphila aggravates acute intestinal inflammation in the Salmonella enterica serovar Typhimurium infection mouse model colonized with a simplified human intestinal microbiota was investigated in this study. To unravel the underlying mechanism that led to the observed phenomenon, the time course of events following the infection was analyzed. In mice colonized with a simplified human intestinal microbiota, Salmonella infection induced clear signs of intestinal inflammation three days post infection. The inflammatory response was similar in mice colonized with A. muciniphila before Salmonella infection. These observations were independent of the time when colonization with the simplified human intestinal microbiota occurred, right after birth or only after weaning, and contradict the previous report.
To find out whether A. muciniphila influences the development of chronic intestinal inflammation in a genetically predisposed host, mono-associated interleukin-10-deficient (Il10-/-) mice, Il10-/- mice dual-associated with A. muciniphila and colitogenic Escherichia coli NC101, as well as Il10-/- mice associated with A. muciniphila and a simplified human intestinal microbiota were compared to the respective mice without A. muciniphila. The data clearly show that in these gnotobiotic Il10-/- mice, A. muciniphila neither induces intestinal inflammation itself nor modulates it after induction by a colitogenic bacterium or by a simplified human intestinal microbiota.
The experiments lead to the conclusion that the promotion of intestinal inflammation is not an intrinsic feature of this bacterium. The results of this study encourage the proposed use of A. muciniphila for the prevention or treatment of metabolic disorders.
Salty taste has evolved to maintain electrolyte homeostasis, serving as a detector for salt containing food. In rodents, salty taste involves at least two transduction mechanisms. One is sensitive to the drug amiloride and specific for Na+, involving epithelial sodium channel (ENaC). A second rodent transduction pathway, which is triggered by various cations, is amiloride insensitive and not almost understood to date. Studies in primates showed amiloride-sensitive as well as amiloride-insensitive gustatory responses to NaCl, implying a role of both salt taste transduction pathways in humans. However, sensory studies in humans point to largely amiloride-insensitive sodium taste perception. An involvement of ENaC in human sodium taste perception was not shown, so far. In this study, ENaC subunit protein and mRNA could be localized to human taste bud cells (TBC). Thus, basolateral αβγ-ENaC ion channels are likely in TBC of circumvallate papillae, possibly mediating basolateral sodium entry. Similarly, basolateral βγ-ENaC might play a role in fungiform TBC. Strikingly, δ-ENaC subunit was confined to taste bud pores of both papillae, likely mediating gustatory sodium entry in TBC, either apical or paracellular via tight junctions. However, regional separation of δ-ENaC and βγ-ENaC in fungiform and circumvallate TBC indicate the presence of unknown interaction partner necessary to assemble into functional ion channels. However, screening of a macaque taste tissue cDNA library did neither reveal polypeptides assembling into a functional cation channel by interaction with δ-ENaC or βγ-ENaC nor ENaC independent salt taste receptor candidates. Thus, ENaC subunits are likely involved in human taste transduction, while exact composition and identity of an amiloride (in)sensitive salt taste receptors remain unclear. Localization of δ-ENaC in human taste pores strongly suggests a role in human taste transduction. In contrast, δ-ENaC is classified as pseudogene Scnn1d in mouse. However, no experimental detected sequences are annotated, while evidences for parts of Scnn1d derived mRNAs exist. In order to elucidate if Scnn1d is possibly involved in rodent salt taste perception, Scnn1d was evaluated in this study to clarify if Scnn1d is a gene or a transcribed pseudogene in mice. Comparative mapping of human SCNN1D to mouse chromosome 4 revealed complete Scnn1d sequence as well as its pseudogenization by Mus specific endogenous retroviruses. Moreover, tissue specific transcription of unitary Scnn1d pseudogene was found in mouse vallate papillae, kidney and testis and led to identification of nine Scnn1d transcripts. In vitro translation experiments showed that Scnn1d transcripts are coding competent for short polypeptides, possibly present in vivo. However, no sodium channel like function or sodium channel modulating activity was evident for Scnn1d transcripts and/or derived polypeptides. Thus, an involvement of mouse δ-ENaC in sodium taste transduction is unlikely and points to species specific differences in salt taste transduction mechanisms.
Homocystein (tHcy) gilt als unabhängiger kardiovaskulärer Risikofaktor und korreliert eng mit einer endothelialen Dysfunktion, welche nichtinvasiv mittels der flussinduzierten Vasodilatation (FMD) messbar ist. Experimentelle Hyperhomocysteinämie ist mit einer reduzierten Bioverfügbarkeit von endothelialen Stickstoffmonoxid (NO) bei gleichzeitig erhöhten Spiegeln des kompetetiven Inhibitors der NO-Biosynthese asymmetrisches Dimethylarginin (ADMA) assoziiert. In-vivo senkt eine Östrogenbehandlung neben tHcy auch die ADMA-Spiegel und verbessert signifikant die Endothelfunktion. Hinsichtlich ihrer Wirkung als selektive Östrogenrezeptormodulatoren wird angenommen, dass Phytoöstrogene, speziell Sojaisoflavone, ähnliche Effekte hervorrufen. Innerhalb einer europäischen, multizentrischen, doppelblinden Interventionsstudie an 89 gesunden, postmenopausalen Frauen wurde der Einfluss von Sojaisoflavonen auf den Homocysteinmetabolismus, den Blutdruck und die in-vivo Endothelfunktion untersucht. Die cross-over Studie umfasste zwei achtwöchige Interventionsperioden, die von einer gleichlangen Wash-out-Phase unterbrochen waren. Die Zuteilung zum Isoflavon- (50 mg/d) oder Plazeboregime für die erste Interventionsphase erfolgte randomisiert. Endpunkterhebungen fanden jeweils in den Wochen 0 und 8 der Interventionsperioden statt. Die renale Ausscheidung von Genistein, Daidzein und Equol war während der Isoflavonintervention signifikant erhöht (P>0,001). Die Phyoöstrogene hatten weder einen Effekt auf die tHcy-Konzentration (P=0,286), noch auf ADMA, Erythrozytenfolat und Vitamin B-12 (P>0,05) im Plasma. Während die Summe aus Nitrat und Nitrit (NOx), welche die NO-Bioverfügbarkeit reflektiert, im Verlaufe der Plazebobehandlung abfiel, wurde ein leichter Anstieg bei der Isoflavonsupplementation beobachtet (Delta Wo8-Wo0: -2,60 [-8,75; 2,25] vs. 1,00 [-6,65; 7,85] µmol/L P<0,001), was zu einem signifikanten Behandlungseffekt führte. Weiterhin wurde eine positive Korrelation zwischen ADMA und Vitamin B-12 gefunden (R=0,252; P=0,018). Die flussinduzierte Vasodilatation (P=0,716), ein Maß für die Endothelfunktion, blieb durch die Isoflavonbehandlung unbeeinflusst, obwohl sich diese über die Zeit insgesamt verbesserte (P>0,001). Bis auf einen marginalen Anstieg des systolischen Wertes (P=0,032) im Vergleich zur Plazebobehandlung blieb der Blutdruck während der Isoflavonintervention unverändert. Im Gegensatz zu Östrogen übten Sojaisoflavone weder einen Einfluss auf die in-vivo Endothelfunktion noch auf die traditionellen und neuen kardiovaskulären Risikofaktoren den Blutdruck, tHcy und ADMA aus. Demzufolge ist der gesundheitliche Nutzen isolierter Isoflavone hinsichtlich einer Prävention hormonmangelbedingter Erkrankungen in gesunden postmenopausalen Frauen fraglich.
Die Abbaubarkeit hochmolekularer Inhaltsstoffe des Kaffeegetränks durch die humane Darmmikrobiota
(2008)
Over the last decades, interest in the impact of the intestinal microbiota on host health has steadily increased. Diet is a major factor that influences the gut microbiota and thereby indirectly affects human health. For example, a high fat diet rich in saturated fatty acids led to an intestinal proliferation of the colitogenic bacterium Bilophila (B.) wadsworthia by stimulating the release of the bile acid taurocholate (TC). TC contains the sulfonated head group taurine, which undergoes conversion to sulfide (H2S) by B. wadsworthia. In a colitis prone murine animal model (IL10 / mice), the bloom of B. wadsworthia was accompanied by an exacerbation of intestinal inflammation. B. wadsworthia is able to convert taurine and also other sulfonates to H2S, indicating the potential association of sulfonate utilization and the stimulation of colitogenic bacteria.
This potential link raised the question, whether dietary sulfonates or their sulfonated metabolites stimulate the growth of colitogenic bacteria such as B. wadsworthia and whether these bacteria convert sulfonates to H2S. Besides taurine, which is present in meat, fish and life-style beverages, other dietary sulfonates are part of daily human nutrition. Sulfolipids such as sulfoquinovosyldiacylglycerols (SQDG) are highly abundant in salad, parsley and the cyanobacterium Arthrospira platensis (Spirulina). Based on previous findings, Escherichia (E.) coli releases the polar headgroup sulfoquinovose (SQ) from SQDG. Moreover, E. coli is able to convert SQ to 2,3 dihydroxypropane 1 sulfonate (DHPS) under anoxic conditions. DHPS is also converted to H2S by B. wadsworthia or by other potentially harmful gut bacteria such as members of the genus Desulfovibrio. However, only few studies report the conversion of sulfonates to H2S by bacteria directly isolated from the human intestinal tract. Most sulfonate utilizing bacteria were obtained from environmental sources such as soil or lake sediment or from potentially intestinal sources such as sewage.
In the present study, fecal slurries from healthy human subjects were incubated with sulfonates under strictly anoxic conditions, using formate and lactate as electron donors. Fecal slurries that converted sulfonates to H2S, were used as a source for the isolation of H2S forming bacteria. Isolates were identified based on their 16S ribosomal RNA (16S rRNA) gene sequence. In addition, conventional C57BL/6 mice were fed a semisynthetic diet supplemented with the SQDG rich Spirulina (SD) or a Spirulina free control diet (CD). During the intervention, body weight, water and food intake were monitored and fecal samples were collected. After three weeks, mice were killed and organ weight and size were measured, intestinal sulfonate concentrations were quantified, gut microbiota composition was determined and parameters of intestinal and hepatic fat metabolism were analyzed.
Human fecal slurries converted taurine, isethionate, cysteate, 3 sulfolacate, SQ and DHPS to H2S. However, inter individual differences in the degradation of these sulfonates were observed. Taurine, isethionate, and 3 sulfolactate were utilized by fecal microbiota of all donors, while SQ, DHPS and cysteate were converted to H2S only by microbiota from certain individuals. Bacterial isolates from human feces able to convert sulfonates to H2S were identified as taurine-utilizing Desulfovibrio strains, taurine- and isethionate-utilizing B. wadsworthia, or as SQ- and 3-sulfolactate- utilizing E. coli. In addition, a co culture of E. coli and B. wadsworthia led to complete degradation of SQ to H2S, with DHPS as an intermediate. Of the human fecal isolates, B. wadsworthia and Desulfovibrio are potentially harmful. E. coli strains might be also pathogenic, but isolated E. coli strains from human feces were identified as commensal gut bacteria.
Feeding SD to mice increased the cecal and fecal SQ concentration and altered the microbiota composition, but the relative abundance of SQDG or SQ converting bacteria and colitogenic bacteria was not enriched in mice fed SD for 21 days. SD did not affect the relative abundance of Enterobacteriaceae, to which the SQDG- and SQ-utilizing E. coli strain belong to. Furthermore, the abundance of B. wadsworthia decreased from day 2 to day 9 in feces, but recovered afterwards in the same mice. In cecum, the family Desulfovibrionaceae, to which B. wadsworthia and Desulfovibrio belong to, were reduced. No changes in the number of B. wadsworthia in cecal contents or of Desulfovibrionaceae in feces were observed. SD led to a mild activation of the immune system, which was not observed in control mice fed CD. Mice fed SD had an increased body weight, a higher adipose tissue weight, and a decreased liver weight compared to the control mice, suggesting an impact of Spirulina supplementation on fat metabolism. However, expression levels of genes involved in intestinal and hepatic intracellular lipid uptake and availability were reduced. Further investigations on the lipid metabolism at protein level could help to clarify these discrepancies.
In summary, humans differ in the ability of their fecal microbiota to utilize dietary sulfonates. While sulfonates stimulated the proliferation of potentially colitogenic isolates from human fecal slurries, the increased availability of SQ in Spirulina fed conventional mice did not lead to an enrichment of such bacteria. Presence or absence of these bacteria may explain the inter individual differences in sulfonate conversion observed for fecal slurries. This work provides new insights in the ability of intestinal bacteria to utilize sulfonates and thus, contributes to a better understanding of microbiota-mediated effects on dietary sulfonate utilization. Interestingly, feeding of the Spirulina-supplemented diet led to body-weight gain in mice in the first two days of intervention, the reasons for which are unknown.
Mycotoxins are secondary metabolites produced by several filamentous fungal species, thus occurring ubiquitously in the environment and food. While the heterogeneous group shows differences in their bioavailability and toxicity, the low-molecular-weight xenobiotics are capable of impacting human and animal health acutely and chronically. Therefore, maximum levels for the major mycotoxins in food and feed are regulated in the current European legislation. Besides free mycotoxins, naturally occurring modified mycotoxins are gaining more attention in recent years. Modified mycotoxins constitute toxins altered by plants, microorganisms, and living organisms in different metabolic pathways or food processing steps. The toxicological relevant compounds often co-occur with their free forms in infested food and feed. Thus, the toxins may contribute to the overall toxicity of mycotoxins, wherefore their presence and toxicity should be considered in risk assessment. Until now, however, there are no regulated limits for modified mycotoxins within the European Union. In this thesis, rapid, sensitive, and robust methods for the analysis of mycotoxins and their modified forms were developed and validated using state-of-the-art high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) systems. Firstly, two analytical methods for determining 38 mycotoxins in cereals and 41 mycotoxins in beer were established since agricultural products count as the primary source of mycotoxin contamination. For the analysis of cereal samples, a QuEChERS- based extraction approach was pursued, while analytes from beer samples were extracted using an acetonitrile precipitation scheme. Validation in cereals, namely wheat, corn, rice, and barley, as well as in beer, demonstrated satisfactory results. To obtain information regarding the natural occurrence of mycotoxins in food products, the developed methods were applied to the analysis of several commercial samples partly produced worldwide. The Fusarium toxins deoxynivalenol and its conjugated metabolite deoxynivalenol-3-glucoside turned out to be the most abundant toxins. None of the other modified mycotoxins were quantified in the samples. However, one cereal sample showed traces of zearalenone- 14-sulfate below the limit of quantification. Moreover, pesticides, plant growth regulators, and tropane alkaloids were investigated in this thesis. Pesticides present biologically highly effective compounds applied in the environment to protect humans from the hazardous effects of pests. While plant growth regulators show similar functions, mainly improving agricultural production, tropane alkaloids are naturally occurring secondary metabolites mainly in the species of Solanaceae that may pose unintended poisoning of humans. The third part of the present thesis aimed to analyze cereal-relevant compounds simultaneously, wherefore a multi-method for the analysis of (modified) mycotoxins, pesticides, plant growth regulators, and tropane alkaloids was established. After processing the samples, this should be done in a single extraction step with subsequent one-time measurements. Various sample preparation procedures were compared, whereby an approach based on an acidified acetonitrile/water extraction, followed by an online clean-up, was finally chosen. The simultaneous determination of more than 350 analytes required an analytical tool that offered an increased resolving power, represented as an enhanced peak capacity, and the possibility of analyzing a broad polarity range. Thus, a two-dimensional LC-MS/MS system based on two different separation mechanisms that performed orthogonal to one another was used for the analysis. Validation of the developed method revealed good performance characteristics for most analytes, while subsequent application showed that 86% of the samples were contaminated with at least one compound. In summary, this thesis provides novel insights into the analysis of food-relevant (modified) mycotoxins. Different sample preparation and LC-MS/MS approaches were introduced, resulting in the development of three new analytical methods. For the first time, such a high number of modified mycotoxins was included in multi-mycotoxin methods and a multi-method ranging both contaminants and residues. Although first steps towards the analysis of modified mycotoxins have been made, further research is needed to elucidate their (co-) occurrence and toxicological behavior in order to understand their relevance to human health in the future.
The trace elements, selenium (Se) and copper (Cu) play an important role in maintaining normal brain function. Since they have essential functions as cofactors of enzymes or structural components of proteins, an optimal supply as well as a well-defined homeostatic regulation are crucial. Disturbances in trace element homeostasis affect the health status and contribute to the incidence and severity of various diseases. The brain in particular is vulnerable to oxidative stress due to its extensive oxygen consumption and high energy turnover, among other factors. As components of a number of antioxidant enzymes, both elements are involved in redox homeostasis. However, high concentrations are also associated with the occurrence of oxidative stress, which can induce cellular damage. Especially high Cu concentrations in some brain areas are associated with the development and progression of neurodegenerative diseases such as Alzheimer's disease (AD). In contrast, reduced Se levels were measured in brains of AD patients. The opposing behavior of Cu and Se renders the study of these two trace elements as well as the interactions between them being particularly relevant and addressed in this work.
Systemic inflammation is a hallmark of cancer cachexia. Among tumor-host interactions, the white adipose tissue (WAT) is an important contributor to inflammation as it suffers morphological reorganization and lipolysis, releasing free fatty acids (FA), bioactive lipid mediators (LM) and pro-inflammatory cytokines, which accentuate the activation of pro-inflammatory signaling pathways and the recruitment of immune cells to the tissue. This project aimed to investigate which inflammatory factors are involved in the local adipose tissue inflammation and what is the influence of such factors upon enzymes involved in FA or LM metabolism in healthy individuals (Control), weight stable gastro-intestinal cancer patients (WSC) and cachectic cancer patients (CC). The results demonstrated that the inflammatory signature of systemic inflammation is different from local adipose tissue inflammation. The systemic inflammation of the cachectic cancer patients was characterized by higher levels of circulating saturated fatty acids (SFA), tumor-necrosis-factor-α (TNF-α), interleukins IL-6, IL-8 and CRP while levels of polyunsaturated fatty acids (PUFAs), especially n3-PUFAs, were lower in CC than in the other groups. In vitro and in adipose tissue explants, pro-inflammatory cytokines and SFAs were shown to increase the chemokines IL-8 and CXCL10 that were found to be augmented in adipose tissue inflammation in CC which was more profound in the visceral adipose tissue (VAT) than in subcutaneous adipose tissue (SAT). Systemic inflammation was negatively associated with the expression of PUFA synthesizing enzymes, though gene and protein expression did hardly differ between groups. The effects of inflammatory factors on enzymes in the whole tissue could have been masked by differentiated modulation of the diverse cell types in the same tissue. In vitro experiments showed that the expression of FA-modifying enzymes such as desaturases and elongases in adipocytes and macrophages was regulated into opposing directions by TNF-α, IL-6, LPS or palmitate. The higher plasma concentration of the pro-resolving LM resolvin D1 in CC cannot compensate the overall inflammatory status and the results indicate that inflammatory cytokines interfere with synthesis pathways of pro-resolving LM. In summary, the data revealed a complex inter-tissue and inter-cellular crosstalk mediated by pro-inflammatory cytokines and lipid compounds enhancing inflammation in cancer cachexia by feed-forward mechanisms.
Background: The role of fatty acid (FA) intake and metabolism in type 2 diabetes (T2D) incidence is controversial. Some FAs are not synthesised endogenously and, therefore, these circulating FAs reflect dietary intake, for example, the trans fatty acids (TFAs), saturated odd chain fatty acids (OCFAs), and linoleic acid, an n-6 polyunsaturated fatty acids (PUFA). It remains unclear if intake of TFA influence T2D risk and whether industrial TFAs (iTFAs) and ruminant TFAs (rTFAs) exert the same effect. Unlike even chain saturated FAs, the OCFAs have been inversely associated with T2D risk, but this association is poorly understood. Furthermore, the associations of n-6 PUFAs intake with T2D risk are still debated, while delta-5 desaturase (D5D), a key enzyme in the metabolism of PUFAs, has been consistently related to T2D risk. To better understand these relationships, the FA composition in circulating lipid fractions can be used as biomarkers of dietary intake and metabolism. The exploration of TFAs subtypes in plasma phospholipids and OCFAs and n-6 PUFAs within a wide range of lipid classes may give insights into the pathophysiology of T2D.
Aim: This thesis aimed mainly to analyse the association of TFAs, OCFAs and n-6 PUFAs with self-reported dietary intake and prospective T2D risk, using seven types of TFAs in plasma phospholipids and deep lipidomics profiling data from fifteen lipid classes.
Methods: A prospective case-cohort study was designed within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study, including all the participants who developed T2D (median follow-up 6.5 years) and a random subsample of the full cohort (subcohort: n=1248; T2D cases: n=820). The main analyses included two lipid profiles. The first was an assessment of seven TFA in plasma phospholipids, with a modified method for analysis of FA with very low abundances. The second lipid profile was derived from a high-throughout lipid profiling technology, which identified 940 distinct molecular species and allowed to quantify OCFAs and PUFAs composition across 15 lipid classes. Delta-5 desaturase (D5D) activity was estimated as 20:4/20:3-ratio. Using multivariable Cox regression models, we examined the associations of TFA subtypes with incident T2D and class-specific associations of OCFA and n-6 PUFAs with T2D risk.
Results: 16:1n-7t, 18:1n-7t, and c9t11-CLA were positively correlated with the intake of fat-rich dairy foods. iTFA 18:1 isomers were positively correlated with margarine. After adjustment for confounders and other TFAs, higher plasma phospholipid concentrations of two rTFAs were associated with a lower incidence of T2D: 18:1n-7t and t10c12-CLA. In contrast, the rTFA c9t11-CLA was associated with a higher incidence of T2D. rTFA 16:1n-7t and iTFAs (18:1n-6t, 18:1n-9t, 18:2n-6,9t) were not statistically significantly associated with T2D risk.
We observed heterogeneous integration of OCFA in different lipid classes, and the contribution of 15:0 versus 17:0 to the total OCFA abundance differed across lipid classes. Consumption of fat-rich dairy and fiber-rich foods were positively and red meat inversely correlated to OCFA abundance in plasma phospholipid classes. In women only, higher abundances of 15:0 in phosphatidylcholines (PC) and diacylglycerols (DG), and 17:0 in PC, lysophosphatidylcholines (LPC), and cholesterol esters (CE) were inversely associated with T2D risk. In men and women, a higher abundance of 15:0 in monoacylglycerols (MG) was also inversely associated with T2D. Conversely, a higher 15:0 concentration in LPC and triacylglycerols (TG) was associated with higher T2D risk in men. Women with a higher concentration of 17:0 as free fatty acids (FFA) also had higher T2D incidence.
The integration of n-6 PUFAs in lipid classes was also heterogeneous. 18:2 was highly abundant in phospholipids (particularly PC), CE, and TG; 20:3 represented a small fraction of FA in most lipid classes, and 20:4 accounted for a large proportion of circulating phosphatidylinositol (PI) and phosphatidylethanolamines (PE). Higher concentrations of 18:2 were inversely associated with T2D risk, especially within DG, TG, and LPC. However, 18:2 as part of MG was positively associated with T2D risk. Higher concentrations of 20:3 in phospholipids (PC, PE, PI), FFA, CE, and MG were linked to higher T2D incidence. 20:4 was unrelated to risk in most lipid classes, except positive associations were observed for 20:4 enriched in FFA and PE. The estimated D5D activities in PC, PE, PI, LPC, and CE were inversely associated with T2D and explained variance of estimated D5D activity by genomic variation in the FADS locus was only substantial in those lipid classes.
Conclusion: The TFAs' conformation is essential in their relationship to diabetes risk, as indicated by plasma rTFA subtypes concentrations having opposite directions of associations with diabetes risk. Plasma OCFA concentration is linked to T2D risk in a lipid class and sex-specific manner. Plasma n-6 PUFA concentrations are associated differently with T2D incidence depending on the specific FA and the lipid class. Overall, these results highlight the complexity of circulating FAs and their heterogeneous association with T2D risk depending on the specific FA structure, lipid class, and sex. My results extend the evidence of the relationship between diet, lipid metabolism, and subsequent T2D risk. In addition, my work generated several potential new biomarkers of dietary intake and prospective T2D risk.
Diabetes is hallmarked by high blood glucose levels, which cause progressive generalised vascular damage, leading to microvascular and macrovascular complications. Diabetes-related complications cause severe and prolonged morbidity and are a major cause of mortality among people with diabetes. Despite increasing attention to risk factors of type 2 diabetes, existing evidence is scarce or inconclusive regarding vascular complications and research investigating both micro- and macrovascular complications is lacking. This thesis aims to contribute to current knowledge by identifying risk factors – mainly related to lifestyle – of vascular complications, addressing methodological limitations of previous literature and providing comparative data between micro- and macrovascular complications.
To address this overall aim, three specific objectives were set. The first was to investigate the effects of diabetes complication burden and lifestyle-related risk factors on the incidence of (further) complications. Studies suggest that diabetes complications are interrelated. However, they have been studied mainly independently of individuals’ complication burden. A five-state time-to-event model was constructed to examine the longitudinal patterns of micro- (kidney disease, neuropathy and retinopathy) and macrovascular complications (myocardial infarction and stroke) and their association with the occurrence of subsequent complications. Applying the same model, the effect of modifiable lifestyle factors, assessed alone and in combination with complication load, on the incidence of diabetes complications was studied. The selected lifestyle factors were body mass index (BMI), waist circumference, smoking status, physical activity, and intake of coffee, red meat, whole grains, and alcohol. Analyses were conducted in a cohort of 1199 participants with incident type 2 diabetes from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam, who were free of vascular complications at diabetes diagnosis. During a median follow-up time of 11.6 years, 96 cases of macrovascular complications (myocardial infarction and stroke) and 383 microvascular complications (kidney disease, neuropathy and retinopathy) were identified. In multivariable-adjusted models, the occurrence of a microvascular complication was associated with a higher incidence of further micro- (Hazard ratio [HR] 1.90; 95% Confidence interval [CI] 0.90, 3.98) and macrovascular complications (HR 4.72; 95% CI 1.25, 17.68), compared with persons without a complication burden. In addition, participants who developed a macrovascular event had a twofold higher risk of future microvascular complications (HR 2.26; 95% CI 1.05, 4.86). The models were adjusted for age, sex, state duration, education, lifestyle, glucose-lowering medication, and pre-existing conditions of hypertension and dyslipidaemia. Smoking was positively associated with macrovascular disease, while an inverse association was observed with higher coffee intake. Whole grain and alcohol intake were inversely associated with microvascular complications, and a U-shaped association was observed for red meat intake. BMI and waist circumference were positively associated with microvascular events. The associations between lifestyle factors and incidence of complications were not modified by concurrent complication burden, except for red meat intake and smoking status, where the associations were attenuated among individuals with a previous complication.
The second objective was to perform an in-depth investigation of the association between BMI and BMI change and risk of micro- and macrovascular complications. There is an ongoing debate on the association between obesity and risk of macrovascular and microvascular outcomes in type 2 diabetes, with studies suggesting a protective effect among people with overweight or obesity. These findings, however, might be limited due to suboptimal control for smoking, pre-existing chronic disease, or short-follow-up. After additional exclusion of persons with cancer history at diabetes onset, the associations between pre-diagnosis BMI and relative annual change between pre- and post-diagnosis BMI and incidence of complications were evaluated in multivariable-adjusted Cox models. The analyses were adjusted for age, sex, education, smoking status and duration, physical activity, alcohol consumption, adherence to the Mediterranean diet, and family history of diabetes and cardiovascular disease (CVD). Among 1083 EPIC-Potsdam participants, 85 macrovascular and 347 microvascular complications were identified during a median follow-up period of 10.8 years. Higher pre-diagnosis BMI was associated with an increased risk of total microvascular complications (HR per 5 kg/m2 1.21; 95% CI 1.07, 1.36), kidney disease (HR 1.39; 95% CI 1.21, 1.60) and neuropathy (HR 1.12; 95% CI 0.96, 1.31); but no association was observed for macrovascular complications (HR 1.05; 95% CI 0.81, 1.36). Effect modification was not evident by sex, smoking status, or age groups. In analyses according to BMI change categories, BMI loss of more than 1% indicated a decreased risk of total microvascular complications (HR 0.62; 95% CI 0.47, 0.80), kidney disease (HR 0.57; 95% CI 0.40, 0.81) and neuropathy (HR 0.73; 95% CI 0.52, 1.03), compared with participants with a stable BMI. No clear association was observed for macrovascular complications (HR 1.04; 95% CI 0.62, 1.74). The impact of BMI gain on diabetes-related vascular disease was less evident. Associations were consistent across strata of age, sex, pre-diagnosis BMI, or medication but appeared stronger among never-smokers than current or former smokers.
The last objective was to evaluate whether individuals with a high-risk profile for diabetes and cardiovascular disease (CVD) also have a greater risk of complications. Within the EPIC-Potsdam study, two accurate prognostic tools were developed, the German Diabetes Risk Score (GDRS) and the CVD Risk Score (CVDRS), which predict the 5-year type 2 diabetes risk and 10-year CVD risk, respectively. Both scores provide a non-clinical and clinical version. Components of the risk scores include age, sex, waist circumference, prevalence of hypertension, family history of diabetes or CVD, lifestyle factors, and clinical factors (only in clinical versions). The association of the risk scores with diabetes complications and their discriminatory performance for complications were assessed. In crude Cox models, both versions of GDRS and CVDRS were positively associated with macrovascular complications and total microvascular complications, kidney disease and neuropathy. Higher GDRS was also associated with an elevated risk of retinopathy. The discrimination of the scores (clinical and non-clinical) was poor for all complications, with the C-index ranging from 0.58 to 0.66 for macrovascular complications and from 0.60 to 0.62 for microvascular complications.
In conclusion, this work illustrates that the risk of complication development among individuals with type 2 diabetes is related to the existing complication load, and attention should be given to regular monitoring for future complications. It underlines the importance of weight management and adherence to healthy lifestyle behaviours, including high intake of whole grains, moderation in red meat and alcohol consumption and avoidance of smoking to prevent major diabetes-associated complications, regardless of complication burden. Risk scores predictive for type 2 diabetes and CVD were related to elevated risks of complications. By optimising several lifestyle and clinical factors, the risk score can be improved and may assist in lowering complication risk.
Vitamin E ist der Überbegriff für 4 Tocopherole (α, β, γ und δ) sowie 4 Tocotrienole (α, β, γ und δ), die als gemeinsames Merkmal ein Chromanolringsystem sowie eine gesättigte (Tocopherole) bzw. ungesättigte (Tocotrienole) Seitenkette aufweisen. Neben ihrer antioxidativen Wirkung (Schutz von Membranen vor Lipidperoxidaton) konnten für einige Vitamin E - Formen auch eine Reihe von hochspezifischen, nicht-antioxidativen Wirkungen in vitro nachgewiesen werden. Meist bleibt jedoch unklar, ob ein solcher Effekt auch in vivo, also im Tiermodel oder direkt im Menschen, gefunden werden kann. In erster Linie müsste hierbei geklärt werden, ob die jeweilige Vitamin E - Form auch bioverfügbar, also in für eine Wirkung ausreichender Konzentration im Organismus vorhanden ist, oder aber vorher eliminiert und ausgeschieden wird. In dieser Doktorarbeit wurden deshalb wichtige Grundlagen zum Abbau der Tocopherole und Tocotrienole erarbeitet. • In HepG2-Zellen konnte der Abbau der Tocotrienole mit Hilfe flüssig- sowie gaschromatographischer Analysemethoden vollständig aufgeklärt werden. Wie sich hierbei ergab, verläuft der Abbau weitgehend in Analogie zum Abbau der Tocopherole über eine durch Cytochrom P450 katalysierte initiale ω-Hydroxylierung mit 5 nachfolgenden β-Oxidationsschritten. • In vitro konnten in HepG2 – Zellen die Abbauraten der verschiedenen Vitamin E - Formen bestimmt werden. Dies nahmen in folgender Reihenfolge zu: α-Tocopherol < γ-Tocopherol < α-Tocotrienol < γ-Tocotrienol. • Wie sich mit Hilfe eines mit Cytochrom P450 hochangereicherten Homogenats aus Rattenlebern ergab, stellt die initiale ω-Hydroxylierung einen geschwindigkeitsbestimmenden Schritt des Abbaus dar: α-Tocopherol wurde weit langsamer hydroxyliert als alle anderen Vitamin E – Formen. • Der unterschiedliche Abbau von α-Tocopherol und γ-Tocotrienol konnte auch im Mäuseversuch in vivo bestätigt werden. Nach Fütterung von Mäusen mit α-Tocopherol wurden nur geringe Mengen von α-Tocopherolmetaboliten im Urin der Mäuse gefunden, während nach Applikation von γ-Tocotrienol hohe Konzentrationen der γ-Tocotrienolmetabolite nachgewiesen wurden. In Plasma und Leber wiederum wurden (dem Futtergehalt entsprechende) hohe α-Tocopherolkonzentrationen entdeckt, während γ-Tocotrienol selbst nach hoher Gabe nicht oder nur in Spuren nachweisbar war. In HepG2 – Zellen konnte gezeigt werden, dass γ-Tocotrienol eine cytotoxische Wirkung auf die Hepatocarcinoma-Zelllinie HepG2 entfalten kann, indem durch die Aktivierung der proteolytischen Caspase 3 die Induktion des programmierten Zelltodes (Apoptose) ausgelöst wird. Abschliessend lässt sich festhalten, dass der Körper lediglich das natürliche α-Tocopherol vor dem Abbau bewahrt, die anderen Vitamin E – Formen jedoch als Fremdstoffe behandelt und rapide ausscheidet. Als doppelter Schutz vor Verlust des “wertvollen” α-Tocopherol dienen hierbei das α-Tocopherol Transfer Protein sowie die in dieser Arbeit gefundenen Unterschiede im ersten Schritt des Abbaus, der Cytochrom P450 - katalysierten ω-Hydroxylierung. Beides erklärt die bevorzugte Retention von α-Tocopherol im Organsimus und seine hohe Bioaktivität. Will man deshalb in vitro Ergebnisse anderer Vitamin E – Formen auf die in vivo Situation übertragen, muss man die geringe Bioverfügbarkeit dieser Substanzen berücksichtigen.
Im Rahmen der EU-weiten REACH-Verordnung haben Alternativmethoden zum Tierversuch in der Toxikologie an Bedeutung gewonnen. Die Alternativmethoden gliedern sich auf in In-vitro- und In-silico-Methoden. In dieser Dissertation wurden verschiedene Konzepte der In-silico-Toxikologie behandelt.
Die bearbeiteten Themen reichen von quantitativen Strukturaktivitätsbeziehungen (QSAR) über eine neue Herangehensweise an das gängige Konzept zur Festlegung von Grenzwerten bis hin zu computerbasierten Modellierungen zum Alkohol- und Bisphenol-A-Stoffwechsel.
Das Kapitel über QSAR befasst sich im Wesentlichen mit der Erstellung und Analyse einer Datenbank mit 878 Substanzen, die sich aus Tierversuchsstudien aus dem Archiv des Bundesinstituts für Risikobewertung zusammensetzt. Das Design wurde dabei an eine bereits bestehende Datenbank angepasst, um so einen möglichst großen Datenpool zu generieren. In der Analyse konnte u.a. gezeigt werden, dass Stoffe mit niedrigerem Molekulargewicht ein erhöhtes Potential für toxikologische Schäden aufwiesen als größere Moleküle.
Mit Hilfe des sogenannten TTC-Konzepts können Grenzwerte für Stoffe geringer Exposition festgelegt werden, zu denen keine toxikologischen Daten zur Verfügung stehen. In dieser Arbeit wurden für die Stoffe dreier Datenbanken entsprechende Grenzwerte festgelegt. Es erfolgte zunächst eine gängige strukturbasierte Aufteilung der Substanzen in die Kategorien "nicht toxisch", "möglicherweise toxisch" und "eindeutig toxisch". Substanzen, die aufgrund ihrer Struktur in eine der drei Klassen eingeordnet werden, erhalten den entsprechenden Grenzwert. Da in die dritte Klasse auch Stoffe eingeordnet werden, deren Toxizität nicht bestimmbar ist, ist sie sehr groß. Daher wurden in dieser Arbeit die ersten beiden Klassen zusammengelgt, um einen größeren Datenpool zu ermöglichen. Eine weitere Neuerung umfasst die Erstellung eines internen Grenzwerts. Diese Vorgehensweise hat den Vorteil, dass der Expositionsweg herausgerechnet wird und somit beispielsweise Studien mit oraler Verabreichung mit Studien dermaler Verabreichung verglichen werden können.
Mittels physiologisch basiertem kinetischem Modelling ist es möglich, Vorgänge im menschlichen Körper mit Hilfe spezieller Software nachzuvollziehen. Durch diese Vorgehensweise können Expositionen von Chemikalien simuliert werden. In einem Teil der Arbeit wurden Alkoholexpositionen von gestillten Neugeborenen simuliert, deren Mütter unmittelbar zuvor alkoholische Getränke konsumiert hatten. Mit dem Modell konnte gezeigt werden, dass die Expositionen des Kindes durchweg gering waren. Nach einem Glas Wein wurden Spitzenkonzentrationen im Blut von Neugeborenen von 0,0034 Promille ermittelt. Zum Vergleich wurde die Exposition durch ein für Säuglinge zugelassenes alkoholhaltiges pflanzliches Arzneimittel simuliert. Hier wurden Spitzenkonzentrationen von 0,0141 Promille erreicht. Daher scheinen Empfehlungen wie gelegentlicher Konsum ohne schädigende Wirkung auf das Kind wissenschaftlich fundiert zu sein.
Ein weiteres Kinetik-Modell befasste sich mit dem Stoffwechsel von Bisphenol A. Teils widersprüchliche Daten zur Belastung mit BPA in der wissenschaftlichen Literatur führen wiederholt zu Anregungen, den Grenzwert der Chemikalie anzupassen. Die Funktionalität der am Metabolismus beteiligten Enzyme kann je nach Individuum unterschiedlich ausgeprägt sein. Mittels Modellings konnte hier gezeigt werden, dass dies maßgeblich dazu führt, dass sich berechnete Plasmaspiegel von Individuen bis zu 4,7-fach unterscheiden.
Die Arbeit konnte somit einen Beitrag zur Nutzung und Weiterentwicklung von In-silico-Modellen für diverse toxikologische Fragestellungen leisten.
Aromatische Amine und Amide (aAA) sind aufgrund ihrer starken Verbreitung in der menschlichen Umwelt und ihres kanzerogenen Potenzials von großer toxikologischer Bedeutung. Die Kanzerogenität der aAA wird durch die Mutagenität hochreaktiver Stoffwechselprodukte vermittelt, die in zwei sequenziellen katalytischen Reaktionen entstehen. Die erste ist meistens eine N-Hydroxylierung, die oft durch Cytochrom P450 1A2 (CYP1A2) katalysiert wird. Daran schließt sich eine O-Konjugation durch Sulfotransferasen (SULT) oder N-Acetyltransferasen (NAT) an. Die Bioaktivierung ist ein kritischer Parameter für die Übertragbarkeit von Ergebnissen aus Tiermodellen auf den Menschen. Rekombinante in vitro Systeme, die fremdstoffmetabolisierende Enzyme verschiedener Spezies exprimieren, ermöglichen die vergleichende Untersuchung der Bioaktivierung im Menschen und in Versuchstieren. Ziel des Projektes war die Aufklärung der Bioaktivierung der aAA durch humane Enzyme. Im Vordergrund stand die Untersuchung der Rolle humaner SULT in diesem Prozess. Es wurden rekombinante in vitro Systeme, konstruiert, die CYP1A2 und SULT des Menschen koexprimieren. SULT-cDNAs wurden in den Säugerzell Expressionsvektor pMPSV kloniert und in Standardindikatorzellen für Mutagenitätsuntersuchungen (V79 Zellen aus dem Chinesischen Hamster) transfiziert. Das Expressionsniveau von CYP1A2 und SULT wurde mittels Immunblotanalyse und radiometrischen Aktivitätsmessungen charakterisiert. In den rekombinanten Zellen wurden vier aAA als Modellsubstanzen (2-Acetylaminofluoren, 2-Aminoanthracen, 3′-Methyl-4-dimethylaminoazobenzol, 2,4-Diaminotoluol) auf ihre Mutagenität am hprt-Locus hin untersucht.Die aAA waren in Zellen, die keine rekombinanten Enzyme oder lediglich CYP1A2 exprimierten, nicht mutagen. In Zellen, die CYP1A2 und SULT der Subfamilie 1A koexprimierten, erzeugten sie bereits in geringen Konzentrationen klare mutagene Effekte (0,3 µM für 2-Acetylaminofluoren und 3′-Methyl-4-dimethylaminoazobenzol; 0,1 µM für 2-Aminoanthracen; 10 µM für 2,4-Diaminotoluol). Die stärkste Aktivierung von 2-Acetylaminofluoren und 3′-Methyl-4-dimethylaminoazobenzol erfolgte in der Zelllinie, die CYP1A2 und SULT1A2 koexprimierte; die stärkste Aktivierung von 2,4-Diaminotoluol und 2-Aminoanthracen erfolgte in der Zelllinie, die CYP1A2 und SULT1A1 koexprimierte. Sowohl SULT1A1 als auch SULT1A2 sind im Menschen genetisch polymorph. Ein unterschiedlich starkes Aktivierungspotenzial der Alloenzyme könnte eine individuell unterschiedliche Suszeptibilität für die durch aAA ausgelöste Kanzerogenese bedingen. In HPRT-Mutationsuntersuchungen mit rekombinanten Zellen zeigten die allelischen Varianten der SULT1A2 starke Unterschiede in ihrem Aktivierungpotenzial. Nur in der Zelllinie, die das Alloenzym SULT1A2*1 mit CYP1A2 koexprimierte, wurde 2-Acetylaminofluoren zum Mutagen aktiviert. Zur Aktivierung von 3′-Methyl-4-dimethylaminoazobenzol waren jedoch sowohl das Alloenzym SULT1A2*1 als auch das Alloenzym SULT1A2*2 in der Lage. Die Alloenzyme der SULT1A1 zeigten ein ähnlich gutes Aktivierungspotenzial für aAA. In früheren Studien wurde gezeigt, dass die SULT1C1 der Ratte eine wichtige Rolle bei der Aktivierung der aAA in dieser Spezies spielt. Dahingegen war die humane SULT1C1 nicht in der Lage die untersuchten aAA zu aktivieren. Die Kenntnis solcher Spezieunterschiede könnte wichtig sein um unterschiedliche Organotropismen aAA in Menschen und Tiermodellen zu erklären, da SULT mit starker Gewebespezifität exprimiert werden und das Expressionsmuster für die einzelnen SULT-Formen in Menschen und Ratten sich stark unterscheidet.
The impact of collagen modifications by methylglyoxal on fibroblast function and the role in aging
(2016)
Im Sinne des Refinements von Tierversuchen sollen alle Bedingungen während der Zucht, der Haltung und des Transports von zu Versuchszwecken gehaltenen Tieren und alle Methoden während des Versuchs so verbessert werden, dass die verwendeten Tiere ein minimales Maß an potentiellem Distress, Schmerzen oder Leiden erfahren. Zudem soll ihr Wohlbefinden durch die Möglichkeit des Auslebens speziesspezifischer Verhaltensweisen und die Anwendung tierschonender Verfahren maximal gefördert werden. Zur Etablierung von Grundsätzen des Refinements sind grundlegende Kenntnisse über die physiologischen Bedürfnisse und Verhaltensansprüche der jeweiligen Spezies unabdingbar. Die Experimentatoren sollten das Normalverhalten der Tiere kennen, um potentielle Verhaltensabweichungen, wie Stereotypien, zu verstehen und interpretieren zu können. Standardisierte Haltungsbedingungen von zu Versuchszwecken gehaltenen Mäusen weichen in diversen Aspekten von der natürlichen Umgebung ab und erfordern eine gewisse Adaptation. Ist ein Tier über einen längeren Zeitraum unfähig, sich an die gegebenen Umstände anzupassen, können abnormale Verhaltensweisen, wie Stereotypien auftreten. Stereotypien werden definiert als Abweichungen vom Normalverhalten, die repetitiv und ohne Abweichungen im Ablauf ausgeführt werden, scheinbar keiner Funktion dienen und der konkreten Umweltsituation nicht immer entsprechen.
Bisher war unklar, in welchem Ausmaß stereotypes Verhalten den metabolischen Phänotyp eines Individuums beeinflusst. Ziel dieser Arbeit war es daher, das stereotype Verhalten der FVB/NJ-Maus erstmals detailliert zu charakterisieren, systematisch zusammenzutragen, welche metabolischen Konsequenzen dieses Verhalten bedingt und wie sich diese auf das Wohlbefinden der Tiere und die Verwendung stereotyper Tiere in Studien mit tierexperimentellem Schwerpunkt auswirken.
Der Versuch begann mit der Charakterisierung der mütterlichen Fürsorge in der Parentalgeneration. Insgesamt wurden 35 Jungtiere der F1-Generation vom Absatz an, über einen Zeitraum von 11 Wochen einzeln gehalten, kontinuierlich beobachtet, bis zum Versuchsende wöchentlich Kotproben gesammelt und das Körpergewicht bestimmt. Zusätzlich erfolgten begleitende Untersuchungen wie Verhaltenstests und die Erfassung der physischen Aktivität und metabolischer Parameter. Anschließend wurden u.a. die zerebralen Serotonin- und Dopamingehalte, fäkale Glucocorticoidlevels, hepatisches Glykogen und muskuläre Glykogen- und Triglyceridlevels bestimmt.
Nahezu unabhängig von der mütterlichen Herkunft entwickelte sich bei mehr als der Hälfte der 35 Jungtiere in der F1-Generation stereotypes Verhalten. Diese Daten deuten darauf hin, dass es keine Anzeichen für das Erlernen oder eine direkte genetische Transmission stereotypen Verhaltens bei der FVB/NJ-Maus gibt. Über den gesamten Beobachtungszeitraum zeichneten sich die stereotypen FVB/NJ-Mäuse durch ein eingeschränktes Verhaltensrepertoire aus. Zu Gunsten der erhöhten Aktivität und des Ausübens stereotypen Verhaltens lebten sie insgesamt weniger andere Verhaltensweisen (Klettern, Graben, Nagen) aus. Darüber hinaus waren Stereotypien sowohl im 24-Stunden Open Field Test als auch in der Messeinrichtung der indirekten Tierkalorimetrie mit einer erhöhten Aktivität und Motilität assoziiert, während die circadiane Rhythmik nicht divergierte. Diese erhöhte körperliche Betätigung spiegelte sich in den niedrigeren Körpergewichtsentwicklungen der stereotypen Tiere wieder. Außerdem unterschieden sich die Körperfett- und Körpermuskelanteile.
Zusammenfassend lässt sich sagen, dass das Ausüben stereotypen Verhaltens zu Differenzen im metabolischen Phänotyp nicht-stereotyper und stereotyper FVB/NJ-Mäuse führt. Im Sinne der „Guten Wissenschaftlichen Praxis“ sollte das zentrale Ziel jedes Wissenschaftlers sein, aussagekräftige und reproduzierbare Daten hervorzubringen. Jedoch können keine validen Resultate von Tieren erzeugt werden, die in Aspekten variieren, die für den vorgesehenen Zweck der Studie nicht berücksichtigt wurden. Deshalb sollten nicht-stereotype und stereotype Individuen nicht innerhalb einer Versuchsgruppe randomisiert werden. Stereotype Tiere demzufolge von geplanten Studien auszuschließen, würde allerdings dem Gebot des zweiten R’s – der Reduction – widersprechen. Um Refinement zu garantieren, sollte der Fokus auf der maximal erreichbaren Prävention stereotypen Verhaltens liegen. Diverse Studien haben bereits gezeigt, dass die Anreicherung der Haltungsumwelt (environmental enrichment) zu einer Senkung der Prävalenz von Stereotypien bei Mäusen führt, dennoch kommen sie weiterhin vor. Daher sollte environmental enrichment zukünftig weniger ein „Kann“, sondern ein „Muss“ sein – oder vielmehr: der Goldstandard. Zudem würde eine profunde phänotypische Charakterisierung dazu beitragen, Mausstämme zu erkennen, die zu Stereotypien neigen und den für den spezifischen Zweck am besten geeigneten Mausstamm zu identifizieren, bevor ein Experiment geplant wird.