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The trace elements zinc and manganese are essential for human health, especially due to their enzymatic and protein stabilizing functions. If these elements are ingested in amounts exceeding the requirements, regulatory processes for maintaining their physiological concentrations (homeostasis) can be disturbed. Those homeostatic dysregulations can cause severe health effects including the emergence of neurodegenerative disorders such as Parkinson’s disease (PD). The concentrations of essential trace elements also change during the aging process. However, the relations of cause and consequence between increased manganese and zinc uptake and its influence on the aging process and the emergence of the aging-associated PD are still rarely understood. This doctoral thesis therefore aimed to investigate the influence of a nutritive zinc and/or manganese oversupply on the metal homeostasis during the aging process. For that, the model organism Caenorhabditis elegans (C. elegans) was applied. This nematode suits well as an aging and PD model due to properties such as its short life cycle and its completely sequenced, genetically amenable genome. Different protocols for the propagation of zinc- and/or manganese-supplemented young, middle-aged and aged C. elegans were established. Therefore, wildtypes, as well as genetically modified worm strains modeling inheritable forms of parkinsonism were applied. To identify homeostatic and neurological alterations, the nematodes were investigated with different methods including the analysis of total metal contents via inductively-coupled plasma tandem mass spectrometry, a specific probe-based method for quantifying labile zinc, survival assays, gene expression analysis as well as fluorescence microscopy for the identification and quantification of dopaminergic neurodegeneration.. During aging, the levels of iron, as well as zinc and manganese increased.. Furthermore, the simultaneous oversupply with zinc and manganese increased the total zinc and manganese contents to a higher extend than the single metal supplementation. In this relation the C. elegans metallothionein 1 (MTL-1) was identified as an important regulator of metal homeostasis. The total zinc content and the concentration of labile zinc were age-dependently, but differently regulated. This elucidates the importance of distinguishing these parameters as two independent biomarkers for the zinc status. Not the metal oversupply, but aging increased the levels of dopaminergic neurodegeneration. Additionally, nearly all these results yielded differences in the aging-dependent regulation of trace element homeostasis between wildtypes and PD models. This confirms that an increased zinc and manganese intake can influence the aging process as well as parkinsonism by altering homeostasis although the underlying mechanisms need to be clarified in further studies.
By using mouse outcross populations in combination with bioinformatic approaches, it was possible to identify and characterize novel genes regulating body weight, fat mass and β-cell function, which all contribute to the pathogenesis of obesity and T2D. In detail, the presented studies identified 1. Ifi202b/IFI16 as adipogenic gene involved in adipocyte commitment, maintenance of white adipocyte identity, fat cell size and the inflammatory state of adipose tissue. 2. Pla2g4a/PLA2G4A as gene linked to increased body weight and fat mass with a higher expression in adipose tissue of obese mice and pigs as well as in obese human subjects. 3. Ifgga2/IRGM as novel regulator of lipophagy protecting from excess hepatic lipid accumulation. 4. Nidd/DBA as a diabetogenic locus containing Kti12, Osbpl9, Ttc39a and Calr4 with differential expression in pancreatic islets and/or genetic variants. 5. miR-31 to be higher expressed in adipose tissue of obese and diabetic mice and humans targeting PPARy and GLUT4 and thereby involved in adipogenesis and insulin signaling. 6. Gjb4 as novel gene triggering the development of T2D by reducing insulin secretion, inducing apoptosis and inhibiting proliferation. The performed studies confirmed the complexity and strong genetic heritability character of obesity and T2D. A high number of genetic variations, each with a small effect, are collectively influencing the degree and severity of the disease. The use of mouse outcross populations is a valid tool for disease gene identification; however, to facilitate and accelerate the process of gene identification the combination of mouse cross data with advanced sequencing resources and the publicly available data sets are essential. The main goal for future studies should be the translation of these novel molecular discoveries to useful treatment therapies. More recently, several classes of novel unimolecular combination therapeutics have emerged with superior efficacy than currently prescribed options and pose the potential to reverse obesity and T2D (Finan et al., 2015). The glucagon-like peptide-1 (GLP-1)- estrogen conjugate, which targets estrogen into cells expressing GLP-1 receptors, was shown to improve energy, glucose and lipid metabolism as well as to reduce food reward (Finan et al., 2012; Schwenk et al., 2014; Vogel et al., 2016). Another possibility is the development of miRNA-based therapeutics to prevent obesity and T2D, such as miRNA mimetics, anti-miRNA oligonucleotides and exosomes loaded with miRNAs (Ji and Guo, 2019; Gottmann et al., 2020). As already described, genome-wide association studies for polygenic obesity and T2D traits in humans have also led to the identification of numerous gene variants with modest effect, most of them having an unknown function (Yazdi et al., 2015). These discoveries resulted in novel animal models and have illuminated new biologic pathways. Therefore, the integration of mouse-human genetic approaches and the utilization of the synergistic effects have the potential to lead to the identification of more genes responsible for common Mendelian forms of obesity and T2D, as well as gene × gene and gene × environment interactions (Yazdi et al., 2015; Ingelsson and McCarthy, 2018). This combination may help to unravel the missing heritability of obesity and T2D, to identify novel drug targets and to design more efficient and personalized obesity prevention and management programs.
The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.
The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.
High-salt (HS) diets have recently been linked to oxidative stress in the brain, a fact that may be a precursor to behavioral changes, such as those involving anxiety-like behavior. However, to the best of our knowledge, no study has evaluated the amygdala redox status after consuming a HS diet in the pre- or postweaning periods. This study aimed to evaluate the amygdala redox status and anxiety-like behaviors in adulthood, after inclusion of HS diet in two periods: preconception, gestation, and lactation (preweaning); and only after weaning (postweaning). Initially, 18 females and 9 male Wistar rats received a standard (n = 9 females and 4 males) or a HS diet (n = 9 females and 5 males) for 120 days. After mating, females continued to receive the aforementioned diets during gestation and lactation. Weaning occurred at 21-day-old Wistar rats and the male offspring were subdivided: control-control (C-C)—offspring of standard diet fed dams who received a standard diet after weaning (n = 9–11), control-HS (C-HS)—offspring of standard diet fed dams who received a HS diet after weaning (n = 9–11), HS-C—offspring of HS diet fed dams who received a standard diet after weaning (n = 9–11), and HS-HS—offspring of HS diet fed dams who received a HS diet after weaning (n = 9–11). At adulthood, the male offspring performed the elevated plus maze and open field tests. At 152-day-old Wistar rats, the offspring were euthanized and the amygdala was removed for redox state analysis. The HS-HS group showed higher locomotion and rearing frequency in the open field test. These results indicate that this group developed hyperactivity. The C-HS group had a higher ratio of entries and time spent in the open arms of the elevated plus maze test in addition to a higher head-dipping frequency. These results suggest less anxiety-like behaviors. In the analysis of the redox state, less activity of antioxidant enzymes and higher levels of the thiobarbituric acid reactive substances (TBARS) in the amygdala were shown in the amygdala of animals that received a high-salt diet regardless of the period (pre- or postweaning). In conclusion, the high-salt diet promoted hyperactivity when administered in the pre- and postweaning periods. In animals that received only in the postweaning period, the addition of salt induced a reduction in anxiety-like behaviors. Also, regardless of the period, salt provided amygdala oxidative stress, which may be linked to the observed behaviors.
Food intake is driven by the need for energy but also by the demand for essential nutrients such as protein. Whereas it was well known how diets high in protein mediate satiety, it remained unclear how diets low in protein induce appetite. Therefore, this thesis aims to contribute to the research area of the detection of restricted dietary protein and adaptive responses.
This thesis provides clear evidence that the liver-derived hormone fibroblast growth factor 21 (FGF21) is an endocrine signal of a dietary protein restriction, with the cellular amino acid sensor general control nonderepressible 2 (GCN2) kinase acting as an upstream regulator of FGF21 during protein restriction. In the brain, FGF21 is mediating the protein-restricted metabolic responses, e.g. increased energy expenditure, food intake, insulin sensitivity, and improved glucose homeostasis. Furthermore, endogenous FGF21 induced by dietary protein or methionine restriction is preventing the onset of type 2 diabetes in the New Zealand Obese mouse.
Overall, FGF21 plays an important role in the detection of protein restriction and macronutrient imbalance in rodents and humans, and mediates both the behavioral and metabolic responses to dietary protein restriction. This makes FGF21 a critical physiological signal of dietary protein restriction, highlighting the important but often overlooked impact of dietary protein on metabolism and eating behavior, independent of dietary energy content.
Fibroblast growth differentiation factor 21 (FGF21) is known as a pivotal regulator of the glucose and lipid metabolism. As such, it is considered beneficial and has even been labelled a longevity hormone. Nevertheless, recent observational studies have shown that FGF21 is increased in higher age with possible negative effects such as loss of lean and bone mass as well as decreased survival. Hepatic FGF21 secretion can be induced by various nutritional stimuli such as starvation, high carbohydrate and fat intake as well as protein deficiency.. So far it is still unclear whether the FGF21 response to different macronutrients is altered in older age. An altered response would potentially contribute to explain the higher FGF21 concentrations found in older age. In this publication-based doctoral dissertation, a cross-sectional study as well as a dietary challenge were conducted to investigate the influence of nutrition on FGF21 concentrations and response in older age. In a cross-sectional study, FGF21 concentrations were assessed in older patients with and without cachexia anorexia syndrome anorexia syndrome compared to an older community-dwelling control group. Cachexia anorexia syndrome is a multifactorial syndrome frequently occurring in old age or in the context of an underlying disease. It is characterized by a severe involuntary weight loss, loss of appetite (anorexia) and reduced food intake, therefore representing a state of severe nutrient deficiency, in some aspects similar to starvation. The highest FGF21 concentrations were found in patients with cachexia anorexia syndrome. Moreover, FGF21 was positively correlated with weight loss and loss of appetite. In addition, cachexia anorexia syndrome itself was associated with FGF21 independent of sex, age and body mass index. As cachectic patients presumably exhibit protein malnutrition and FGF21 has been proposed a marker for protein insufficiency, the higher levels of FGF21 in patients with cachexia anorexia syndrome might be partly explained by insufficient protein intake. In order to investigate the acute response of FGF21 to different nutritional stimuli, a dietary challenge with a parallel group design was conducted. Here, healthy older (65-85 years) and younger (18-35 years) adults were randomized to one of four test meals: a dextrose drink, a high carbohydrate, high fat or high protein meal. Over the course of four hours, postprandial FGF21 concentrations (dynamics) were assessed and the FGF21 response (incremental area under the curve) to each test meal was examined.. In a sub-group of older and younger women, also the adiponectin response was investigated, as adiponectin is a known mediator of FGF21 effects on glucose and lipid metabolism. The dietary meal challenge revealed that dextrose and high carbohydrate intake result in higher FGF21 concentrations after four hours in older adults. This was partly explained by higher postprandial glucose concentrations in the old. For high fat ingestion no age differences were found. For the first time, acute FGF21 response to high protein intake was shown. Here, protein ingestion resulted in lower FGF21 concentrations in younger compared to older adults. Furthermore, sufficient protein intake, according to age-dependent recommendations, of the previous day, was associated with lower FGF21 concentrations in both age groups. The higher FGF21 response to dextrose ingestion resulted in a higher adiponectin response in older women, independent of fat mass, insulin resistance, triglyceride concentrations, inflammation and oxidative stress. Following the high fat meal, adiponectin concentrations declined in older women. Adiponectin response was not affected by meal composition in younger women. In summary, this thesis showed a positive association of FGF21 and cachexia anorexia syndrome with concomitant anorexia in older patients. Regarding the acute FGF21 response, a higher response following dextrose and carbohydrate ingestion was found in older compared with younger subjects. This might be attributed to a higher glucose response in older age. Furthermore, it was shown that the higher FGF21 response after dextrose ingestion possibly contributes to a higher adiponectin response in older women, independent of potential metabolic and inflammatory confounders. Acute protein ingestion resulted in a significant decrease in FGF21 concentrations. Moreover, protein intake of the previous day was inversely associated with fasting FGF21 concentrations. This might explain why FGF21 concentrations are higher in cachexia anorexia syndrome. These results therefore support the role of FGF21 as a sensor of protein restriction.
Over the last few decades, the prevalence of obesity has risen to epidemic proportions worldwide. Consequently, the number of obesity in pregnancy has risen drastically. Gestational overweight and obesity are associated with impaired outcomes for mother and child. Furthermore, studies show that maternal obesity can lead to long-term consequences in the offspring, increasing the risk for obesity and cardiometabolic disease in later life. In addition to genetic mechanisms, mounting evidence demonstrates the induction of epigenetic alterations by maternal obesity, which can affect the offspring's phenotype, thereby influencing the later risk of obesity and cardiometabolic disease. Clear evidence in this regard comes from various animal models of maternal obesity. Evidence derived from clinical studies remains limited. The current article gives an overview of pathophysiological changes associated with maternal obesity and their consequences on placental structure and function. Furthermore, a short excurse is given on epigenetic mechanisms and emerging data regarding a putative interaction between metabolism and epigenetics. Finally, a summary of important findings of animal and clinical studies investigating maternal obesity-related epigenetic effects is presented also addressing current limitations of clinical studies.
The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.
The mitochondrial chaperone complex HSP60/HSP10 facilitates mitochondrial protein homeostasis by folding more than 300 mitochondrial matrix proteins. It has been shown previously that HSP60 is downregulated in brains of type 2 diabetic (T2D) mice and patients,
causing mitochondrial dysfunction and insulin resistance. As HSP60 is also decreased in peripheral tissues in T2D animals, this thesis investigated the effect of overall reduced HSP60 in the development of obesity and associated co-morbidities.
To this end, both female and male C57Bl/6N control (i.e. without further alterations in their genome, Ctrl) and heterozygous whole-body Hsp60 knock-out (Hsp60+/-) mice, which exhibit a 50 % reduction of HSP60 in all tissues, were fed a normal chow diet (NCD) or a highfat diet (HFD, 60 % calories from fat) for 16 weeks and were subjected to extensive metabolic phenotyping including indirect calorimetry, NMR spectroscopy, insulin, glucose and pyruvate tolerance tests, vena cava insulin injections, as well as histological and molecular analysis.
Interestingly, NCD feeding did not result in any striking phenotype, only a mild increase in energy expenditure in Hsp60+/- mice. Exposing mice to a HFD however revealed an increased body weight due to higher muscle mass in female Hsp60+/- mice, with a simultaneous decrease in energy expenditure. Additionally, these mice displayed decreased fasting glycemia. Opposingly, male Hsp60+/- compared to control mice showed lower body weight gain due to decreased fat mass and an increased energy expenditure, strikingly independent of lean mass. Further, only male Hsp60+/- mice display improved HOMA-IR and Matsuda
insulin sensitivity indices.
Despite the opposite phenotype in regards to body weight development, Hsp60+/- mice of both sexes show a significantly higher cell number, as well as a reduction in adipocyte size in the subcutaneous and gonadal white adipose tissue (sc/gWAT). Curiously, this adipocyte hyperplasia – usually associated with positive aspects of WAT function – is disconnected from metabolic improvements, as the gWAT of male Hsp60+/- mice shows mitochondrial dysfunction, oxidative stress, and insulin resistance. Transcriptomic analysis of gWAT shows an up
regulation of genes involved in macroautophagy. Confirmatory, expression of microtubuleassociated protein 1A/1B light chain 3B (LC3), as a protein marker of autophagy, and direct measurement of lysosomal activity is increased in the gWAT of male Hsp60+/- mice.
In summary, this thesis revealed a novel gene-nutrient interaction. The reduction of the crucial chaperone HSP60 did not have large effects in mice fed a NCD, but impacted metabolism during DIO in a sex-specific manner, where, despite opposing body weight and
body composition phenotypes, both female and male Hsp60+/- mice show signs of protection from high fat diet-induced systemic insulin resistance.
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.
The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.
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.
A fast high performance liquid chromatography tandem mass spectrometry multi-method based on an ACN-precipitation extraction was developed for the analysis of 41 (modified) mycotoxins in beer. Validation according to the performance criteria defined by the European Commission (EC) in Commission Decision no. 657/2002 revealed good linearity (R2 > 0.99), repeatability (RSDr < 15%), reproducibility (RSDR < 15%), and recovery (79–100%). Limits of quantification ranging from 0.04 to 75 µg/L were obtained. Matrix effects varied from −67 to +319% and were compensated for using standard addition. In total, 87 beer samples, produced worldwide, were analyzed for the presence of mycotoxins with a focus on modified mycotoxins, whereof 76% of the samples were contaminated with at least one mycotoxin. The most prevalent mycotoxins were deoxynivalenol-3-glucoside (63%), HT-2 toxin (15%), and tenuazonic acid (13%). Exposure estimates of deoxynivalenol and its metabolites for German beer revealed no significant contribution to intake of deoxynivalenol.
High-salt (HS) diets have recently been linked to oxidative stress in the brain, a fact that may be a precursor to behavioral changes, such as those involving anxiety-like behavior. However, to the best of our knowledge, no study has evaluated the amygdala redox status after consuming a HS diet in the pre- or postweaning periods. This study aimed to evaluate the amygdala redox status and anxiety-like behaviors in adulthood, after inclusion of HS diet in two periods: preconception, gestation, and lactation (preweaning); and only after weaning (postweaning). Initially, 18 females and 9 male Wistar rats received a standard (n = 9 females and 4 males) or a HS diet (n = 9 females and 5 males) for 120 days. After mating, females continued to receive the aforementioned diets during gestation and lactation. Weaning occurred at 21-day-old Wistar rats and the male offspring were subdivided: control-control (C-C)—offspring of standard diet fed dams who received a standard diet after weaning (n = 9–11), control-HS (C-HS)—offspring of standard diet fed dams who received a HS diet after weaning (n = 9–11), HS-C—offspring of HS diet fed dams who received a standard diet after weaning (n = 9–11), and HS-HS—offspring of HS diet fed dams who received a HS diet after weaning (n = 9–11). At adulthood, the male offspring performed the elevated plus maze and open field tests. At 152-day-old Wistar rats, the offspring were euthanized and the amygdala was removed for redox state analysis. The HS-HS group showed higher locomotion and rearing frequency in the open field test. These results indicate that this group developed hyperactivity. The C-HS group had a higher ratio of entries and time spent in the open arms of the elevated plus maze test in addition to a higher head-dipping frequency. These results suggest less anxiety-like behaviors. In the analysis of the redox state, less activity of antioxidant enzymes and higher levels of the thiobarbituric acid reactive substances (TBARS) in the amygdala were shown in the amygdala of animals that received a high-salt diet regardless of the period (pre- or postweaning). In conclusion, the high-salt diet promoted hyperactivity when administered in the pre- and postweaning periods. In animals that received only in the postweaning period, the addition of salt induced a reduction in anxiety-like behaviors. Also, regardless of the period, salt provided amygdala oxidative stress, which may be linked to the observed behaviors.
The objective of this work was to investigate the potential effect of cereal α-amylase/trypsin inhibitors (ATIs) on growth parameters and selective digestive enzymes of Tenebrio molitor L. larvae. The approach consisted of feeding the larvae with wheat, sorghum and rice meals containing different levels and composition of α-amylase/trypsin inhibitors. The developmental and biochemical characteristics of the larvae were assessed over feeding periods of 5 h, 5 days and 10 days, and the relative abundance of α-amylase and selected proteases in larvae were determined using liquid chromatography tandem mass spectrometry. Overall, weight gains ranged from 21% to 42% after five days of feeding. The larval death rate significantly increased in all groups after 10 days of feeding (p < 0.05), whereas the pupation rate was about 25% among larvae fed with rice (Oryza sativa L.) and Siyazan/Esperya wheat meals, and only 8% and 14% among those fed with Damougari and S35 sorghum meals. As determined using the Lowry method, the protein contents of the sodium phosphate extracts ranged from 7.80 ± 0.09 to 9.42 ± 0.19 mg/mL and those of the ammonium bicarbonate/urea reached 19.78 ± 0.16 to 37.47 ± 1.38 mg/mL. The total protein contents of the larvae according to the Kjeldahl method ranged from 44.0 and 49.9 g/100 g. The relative abundance of α-amylase, CLIP domain-containing serine protease, modular serine protease zymogen and C1 family cathepsin significantly decreased in the larvae, whereas dipeptidylpeptidase I and chymotrypsin increased within the first hours after feeding (p < 0.05). Trypsin content was found to be constant independently of time or feed material. Finally, based on the results we obtained, it was difficult to substantively draw conclusions on the likely effects of meal ATI composition on larval developmental characteristics, but their effects on the digestive enzyme expression remain relevant.
The detection and quantification of nut allergens remains a major challenge. The liquid chroma-tography tandem mass spectrometry (LC-MS/MS) is emerging as one of the most widely used methods, but sample preparation prior to the analysis is still a key issue. The objective of this work was to establish optimized protocols for extraction, tryptic digestion and LC-MS analysis of almond, cashew, hazelnut, peanut, pistachio and walnut samples. Ammonium bicar-bonate/urea extraction (Ambi/urea), SDS buffer extraction (SDS), polyvinylpolypyrroli-done (PVPP) extraction, trichloroacetic acid/acetone extraction (TCA/acetone) and chloro-form/methanol/sodium chloride precipitation (CM/NaCl) as well as the performances of con-ventional tryptic digestion and microwave-assisted breakdown were investigated. Overall, the protein extraction yields ranged from 14.9 ± 0.5 (almond extract from CM/NaCl) to 76.5 ± 1.3% (hazelnut extract from Ambi/urea). Electrophoretic profiling showed that the SDS extraction method clearly presented a high amount of extracted proteins in the range of 0–15 kDa, 15–35 kDa, 35–70 kDa and 70–250 kDa compared to the other methods. The linearity of the LC-MS methods in the range of 0 to 0.4 µg equivalent defatted nut flour was assessed and recovery of internal standards GWGG and DPLNV(d8)LKPR ranged from 80 to 120%. The identified bi-omarkers peptides were used to relatively quantifier selected allergenic protein form the inves-tigated nut samples. Considering the overall results, it can be concluded that SDS buffer allows a better protein extraction from almond, peanut and walnut samples while PVPP buffer is more appropriate for cashew, pistachio and hazelnut samples. It was also found that conventional overnight digestion is indicated for cashew, pistachio and hazelnut samples, while microwave assisted tryptic digestion is recommended for almond, hazelnut and peanut extracts.
Macrophages in pathologically expanded dysfunctional white adipose tissue are exposed to a mix of potential modulators of inflammatory response, including fatty acids released from insulin-resistant adipocytes, increased levels of insulin produced to compensate insulin resistance, and prostaglandin E₂ (PGE₂) released from activated macrophages. The current study addressed the question of how palmitate might interact with insulin or PGE₂ to induce the formation of the chemotactic pro-inflammatory cytokine interleukin-8 (IL-8). Human THP-1 cells were differentiated into macrophages. In these macrophages, palmitate induced IL-8 formation. Insulin enhanced the induction of IL-8 formation by palmitate as well as the palmitate-dependent stimulation of PGE₂ synthesis. PGE₂ in turn elicited IL-8 formation on its own and enhanced the induction of IL-8 release by palmitate, most likely by activating the EP4 receptor. Since IL-8 causes insulin resistance and fosters inflammation, the increase in palmitate-induced IL-8 formation that is caused by hyperinsulinemia and locally produced PGE₂ in chronically inflamed adipose tissue might favor disease progression in a vicious feed-forward cycle.
Macrophages in pathologically expanded dysfunctional white adipose tissue are exposed to a mix of potential modulators of inflammatory response, including fatty acids released from insulin-resistant adipocytes, increased levels of insulin produced to compensate insulin resistance, and prostaglandin E₂ (PGE₂) released from activated macrophages. The current study addressed the question of how palmitate might interact with insulin or PGE₂ to induce the formation of the chemotactic pro-inflammatory cytokine interleukin-8 (IL-8). Human THP-1 cells were differentiated into macrophages. In these macrophages, palmitate induced IL-8 formation. Insulin enhanced the induction of IL-8 formation by palmitate as well as the palmitate-dependent stimulation of PGE₂ synthesis. PGE₂ in turn elicited IL-8 formation on its own and enhanced the induction of IL-8 release by palmitate, most likely by activating the EP4 receptor. Since IL-8 causes insulin resistance and fosters inflammation, the increase in palmitate-induced IL-8 formation that is caused by hyperinsulinemia and locally produced PGE₂ in chronically inflamed adipose tissue might favor disease progression in a vicious feed-forward cycle.