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Human and murine studies identified the lysosomal enzyme acid sphingomyelinase (ASM) as a target for antidepressant therapy and revealed its role in the pathophysiology of major depression. In this study, we generated a mouse model with overexpression of Asm (Asm-tg(fb)) that is restricted to the forebrain to rule out any systemic effects of Asm overexpression on depressive-like symptoms. The increase in Asm activity was higher in male Asm-tg(fb) mice than in female Asm-tg(fb) mice due to the breeding strategy, which allows for the generation of wild-type littermates as appropriate controls. Asm overexpression in the forebrain of male mice resulted in a depressive-like phenotype, whereas in female mice, Asm overexpression resulted in a social anxiogenic-like phenotype. Ceramides in male Asm-tg(fb) mice were elevated specifically in the dorsal hippocampus. mRNA expression analyses indicated that the increase in Asm activity affected other ceramide-generating pathways, which might help to balance ceramide levels in cortical brain regions. This forebrain-specific mouse model offers a novel tool for dissecting the molecular mechanisms that play a role in the pathophysiology of major depression.
Adipogenesis is governed by a well-documented cascade of transcription factors. However, less is known about non-transcription factors that govern early stages of adipogenesis. Here we show that cellular retinol-binding protein type I (CRBP-I), a small cytosolic binding protein for retinol and retinaldehyde, is specifically restricted to preadipocytes in white adipose tissue. The absence of CRBP-I in mice (CRBP-I-KO mice) leads to increased adiposity. Despite increased adiposity, CRBP-I-KO mice remain more glucose tolerant and insulin sensitive during high-fat-diet feeding. 3T3-L1 cells deficient in CRBP-I or mouse embryonic fibroblasts derived from CRBP-I-KO mice had increased adipocyte differentiation and triglyceride (TG) accumulation. This was due to increased expression and activity of PPAR gamma, while other transcription factor pathways in early and late differentiation remained unchanged. Conversely, the overexpression of CRBP-I in 3T3-L1 cells results in decreased TG accumulation. In conclusion, CRBP-I is a cytosolic protein specifically expressed in preadipocytes that regulates adipocyte differentiation in part by affecting PPAR gamma activity.
CXCL12-CXCR4 signaling controls multiple physiological processes and its dysregulation is associated with cancers and inflammatory diseases. To discover as-yet-unknown endogenous ligands of CXCR4, we screened a blood-derived peptide library for inhibitors of CXCR4-tropic HIV-1 strains. This approach identified a 16 amino acid fragment of serum albumin as an effective and highly specific CXCR4 antagonist. The endogenous peptide, termed EPI-X4, is evolutionarily conserved and generated from the highly abundant albumin precursor by pH-regulated proteases. EPI-X4 forms an unusual lasso-like structure and antagonizes CXCL12-induced tumor cell migration, mobilizes stem cells, and suppresses inflammatory responses in mice. Furthermore, the peptide is abundant in the urine of patients with inflammatory kidney diseases and may serve as a biomarker. Our results identify EPI-X4 as a key regulator of CXCR4 signaling and introduce proteolysis of an abundant precursor protein as an alternative concept for chemokine receptor regulation.
Camellia sinensis synthesizes and emits a large variety of volatile phenylpropanoids and benzenoids (VPB). To investigate the enzymes involved in the formation of these VPB compounds, a new C. sinensis short-chain dehydrogenase/reductase (CsSDR) was isolated, cloned, sequenced, and functionally characterized. The complete open reading frame of CsSDR contains 996 nucleotides with a calculated protein molecular mass of 34.5 kDa. The CsSDR recombinant protein produced in Escherichia coli exhibited dehydrogenase-reductase activity towards several major VPB compounds in C. sinensis flowers with a strong preference for NADP/NADPH co-factors, and showed affinity for (R)/(S)-1-phenylethanol (1PE), phenylacetaldehyde, benzaldehyde, and benzyl alcohol, and no affinity for acetophenone (AP) and 2-phenylethanol. CsSDR showed the highest catalytic efficiency towards (R)/(S)-1PE. Furthermore, the transient expression analysis in Nicotiana benthamiana plants validated that CsSDR could convert 1PE to AP in plants. CsSDR transcript level was not significantly affected by floral development and some jasmonic acid-related environmental stress, and CsSDR transcript accumulation was detected in most floral tissues such as receptacle and anther, which were main storage locations of VPB compounds. Our results indicate that CsSDR is expressed in C. sinensis flowers and is likely to contribute to a number of floral VPB compounds including the 1PE derivative AP.
The physiological functions of sphingolipids in animals have been intensively studied, while less attention has been paid to their roles in plants. Here, we reveal the involvement of sphingolipid delta8 desaturase (SlSLD) in the chilling resistance of tomato (Solanum lycopersicum cv. Micro-Tom). We used the virus-induced gene silencing (VIGS) approach to knock-down SlSLD expression in tomato leaves, and then evaluated chilling resistance. Changes in leaf cell structure under a chilling treatment were observed by transmission electron microscopy. In control plants, SlSLD was highly expressed in the fruit and leaves in response to a chilling treatment. The degree of chilling damage was greater in SlSLD-silenced plants than in control plants, indicating that SlSLD knock-down significantly reduced the chilling resistance of tomato. Compared with control plants, SlSLD-silenced plants showed higher relative electrolytic leakage and malondialdehyde content, and lower superoxide dismutase and peroxidase activities after a chilling treatment. Chilling severely damaged the chloroplasts in SlSLD-silenced plants, resulting in the disruption of chloroplast membranes, swelling of thylakoids, and reduced granal stacking. Together, these results show that SlSLD is crucial for chilling resistance in tomato.
Nanomaterials play an important role in mimicking the biochemical and biophysical cues of the extracellular matrix in human mesenchymal stem cells (MSCs). Increasing studies have demonstrated the crucial impact of functional groups on MSCs, while limited research is available on how the functional group's density on nanoparticles regulates MSC behavior. Herein, the effects of dendritic polyglycerol (dPG)-conjugated gold nanostars (GNSs) with different densities of functional groups on the osteogenesis of MSCs are systematically investigated. dPG@GNS nanocomposites have good biocompatibility and the uptake by MSCs is in a functional group density-dependent manner. The osteogenic differentiation of MSCs is promoted by all dPG@GNS nanocomposites, in terms of alkaline phosphatase activity, calcium deposition, and expression of osteogenic protein and genes. Interestingly, the dPGOH@GNSs exhibit a slight upregulation in the expression of osteogenic markers, while the different charged densities of sulfate and amino groups show more efficacy in the promotion of osteogenesis. Meanwhile, the sulfated nanostars dPGS20@GNSs show the highest enhancement. Furthermore, various dPG@GNS nanocomposites exerted their effects by regulating the activation of Yes-associated protein (YAP) to affect osteogenic differentiation. These results indicate that dPG@GNS nanocomposites have functional group density-dependent influence on the osteogenesis of MSCs, which may provide a new insight into regulating stem cell fate.
OBJECTIVE: Higher plasma vitamin C levels are associated with lower type 2 diabetes risk, but whether this association is causal is uncertain. To investigate this, we studied the association of genetically predicted plasma vitamin C with type 2 diabetes.
RESEARCH DESIGN AND METHODS: We conducted genome-wide association studies of plasma vitamin C among 52,018 individuals of European ancestry to discover novel genetic variants. We performed Mendelian randomization analyses to estimate the association of genetically predicted differences in plasma vitamin C with type 2 diabetes in up to 80,983 case participants and 842,909 noncase participants. We compared this estimate with the observational association between plasma vitamin C and incident type 2 diabetes, including 8,133 case participants and 11,073 noncase participants.
RESULTS: We identified 11 genomic regions associated with plasma vitamin C (P < 5 x 10(-8)), with the strongest signal at SLC23A1, and 10 novel genetic loci including SLC23A3, CHPT1, BCAS3, SNRPF, RER1, MAF, GSTA5, RGS14, AKT1, and FADS1. Plasma vitamin C was inversely associated with type 2 diabetes (hazard ratio per SD 0.88; 95% CI 0.82, 0.94), but there was no association between genetically predicted plasma vitamin C (excluding FADS1 variant due to its apparent pleiotropic effect) and type 2 diabetes (1.03; 95% CI 0.96, 1.10).
CONCLUSIONS: These findings indicate discordance between biochemically measured and genetically predicted plasma vitamin C levels in the association with type 2 diabetes among European populations. The null Mendelian randomization findings provide no strong evidence to suggest the use of vitamin C supplementation for type 2 diabetes prevention.
Background and objective Whether treatment with vitamin D receptor activators contributes to cardiovascular disease in patients with chronic kidney disease is a matter of debate. We studied mechanisms involved in vitamin D-related vascular calcifications in vivo and in vitro.
Methods Aortic calcifications were induced in subtotally nephrectomized (SNX) rats by treatment with a high dose (0.25 mu g/kg per day) of 1,25-dihydroxyvitamin D-3 (calcitriol) given for 6 weeks. Likewise, primary rat vascular smooth muscle cells (VSMCs) were incubated with calcitriol at concentrations ranging from 10(-11) to 10(-7) mol/l. Immunohistochemistry revealed that the aortic expression of osteopontin, osteocalcin and bone sialoprotein was significantly increased in calcitriol-treated SNX rats compared to untreated SNX controls. In addition, aortic expression of the transient receptor potential vanilloid calcium channel 6 (TRPV6) and calbindin D9k was significantly up-regulated by treatment with calcitriol. Furthermore, calcitriol significantly increased expression of the osteogenic transcription factor osterix. In-vitro studies showed similar results, confirming that these effects could be attributed to treatment with calcitriol.
Conclusions High-dose calcitriol treatment induces an osteoblastic phenotype in VSMC both in SNX rats and in vitro, associated with up-regulation of proteins regulating mineralization and calcium transport, and of the osteogenic transcription factor osterix.
Aims:This study aims to investigate the relationship between plasma endothelin-1 (ET-1) concentrations, ET-1 gene polymorphisms in loci rs5370, rs1630736, 3A/4A and clinical features of primary nephrotic syndrome (NS) in children.
Materials and methods: Thirty-six children with primary NS were selected as case group, and 94 healthy children were selected as control group. All subjects were genotyped for three single nucleotide polymorphisms (SNPs) (rs5370, rs10478694 [3A4A) and rs 1630736) in the ET-1 gene by gene sequencing. The plasma ET-1 concentrations were measured using a radio-immunoassay.
Key findings: Plasma ET-1 concentrations were higher in NS patients (P = 0.007) as compared to healthy children. The allele frequencies between control and NS patients were significantly different only with respect to the rs10478694 SNP of the ET-1 gene. The allele frequencies between control and NS patients for the rs5370 SNP showed a trend towards difference (P = 0.057). Plasma cholesterol in NS patients is associated with both: the Cl genotype in locus rs5370 and the 3A4A genotype in locus rs10478694 (P < 0.05 in both cases).
Significance: The ET systems might play a disease modifying role in pediatric NS. Plasma cholesterol, a hallmark of NS. seems to be associated with genetic variations within the human ET-1 gene. (C) 2014 Elsevier Inc. All rights reserved.
Green algae are of great economic importance. Seaweed is consumed fresh or as seasoning in Japan. The commercial value is determined by quality, color, and flavor and is also strongly influenced by the production area. Our research, based on solid phase microextraction gas chromatography mass spectrometry (SPME-GC-MS), has revealed that volatile compounds differ intensely in the four varieties of commercial green algae. Accordingly, 41 major volatile compounds were identified. Heptadecene was the most abundant compound from Okayama (Ulva prolifera), Tokushima (Ulva prolifera), and Ehime prefecture (Ulva linza). Apocarotenoids, such as ionones, and their derivatives were prominent volatiles in algae from Okayama (Ulva prolifera) and Tokushima prefecture (Ulva prolifera). Volatile, short chained apocarotenoids are among the most potent flavor components and contribute to the flavor of fresh, processed algae, and algae-based products. Benzaldehyde was predominant in seaweed from Shizuoka prefecture (Monostroma nitidum). Multivariant statistical analysis (PCA) enabled simple discrimination of the samples based on their volatile profiles. This work shows the potential of SPME-GC-MS coupled with multivariant analysis to discriminate between samples of different geographical and botanical origins and form the basis for development of authentication methods of green algae products, including seasonings.
In nature, plants interact with numerous beneficial or pathogenic soil-borne microorganisms. Plants have developed various defense strategies to expel pathogenic microbes, some of which function soon after pathogen infection. We used Medicago truncatula and its oomycete pathogen Aphanomyces euteiches to elucidate early responses of the infected root. A. euteiches causes root rot disease in legumes and is a limiting factor in legume production. Transcript profiling of seedlings and adult plant roots inoculated with A. euteiches zoospores for 2 h revealed specific upregulation of a gene encoding a putative sesquiterpene synthase (M. truncatula TERPENE SYNTHASE 10 [MtTPS10]) in both developmental stages. MtTPS10 was specifically expressed in roots upon oomycete infection. Heterologous expression of MtTPS10 in yeast led to production of a blend of sesquiterpenes and sesquiterpene alcohols, with NMR identifying a major peak corresponding to himalachol. Moreover, plants carrying a tobacco (Nicotiana tabacum) retrotransposon Tnt1 insertion in MtTPS10 lacked the emission of sesquiterpenes upon A. euteiches infection, supporting the assumption that the identified gene encodes a multiproduct sesquiterpene synthase. Mttps10 plants and plants with reduced MtTPS10 transcript levels created by expression of an MtTPS10-artificial microRNA in roots were more susceptible to A. euteiches infection than were the corresponding wild-type plants and roots transformed with the empty vector, respectively. Sesquiterpenes produced by expression of MtTPS10 in yeast also inhibited mycelial growth and A. euteiches zoospore germination. These data suggest that sesquiterpene production in roots by MtTPS10 plays a previously unrecognized role in the defense response of M. truncatula against A. euteiches.
Background/Aims: A recent study revealed that global overexpression of ET-1 causes a slight reduction in systemic blood pressure. Moreover, heterozygous ET-1 knockout mice are hypertensive. The role of ET-1 in human hypertension was so far not addressed by a strict meta-analysis of published human clinical studies.
Methods: We included studies published between January 1, 1990 and February 28, 2017. We included case control studies analyzing untreated essential hypertension or hypertensive patients where antihypertensive medication was discontinued for at least two weeks. Based on the principle of Cochrane systematic reviews, case control studies (CCSs) in PubMed (Medline) and Google Scholar designed to identify the role of endothelin-1 (ET-1) in the pathophysiological of hypertension were screened. Review Manager Version 5.0 (Rev-Man 5.0) was applied for statistical analysis. Mean difference and 95% confidence interval (CI) were shown in inverse variance (IV) fixed-effects model or IV random-effects models.
Results: Eleven studies fulfilling our in-and exclusion criteria were eligible for this meta-analysis. These studies included 450 hypertensive patients and 328 controls. Our meta-analysis revealed that ET-1 plasma concentrations were higher in hypertensive patients as compared to the control patients [mean difference between groups 1.57 pg/mL, 95%Ci [0.47 similar to 2.68, P = 0.005]. These finding were driven by patients having systolic blood pressure higher than 160 mmHg and diastolic blood pressure higher than 100 mmHg.
Conclusions: This meta-analysis showed that hypertensive patients do have elevated plasma ET-1 concentrations. This finding is driven by those patients with high systolic/diastolic blood pressure. Given that the ET-1 gene did not appear in any of the whole genome association studies searching for hypertension associated gene loci, it is very likely that the elevated plasma ET-1 concentrations in hypertensive patients are secondary to hypertension and may reflect endothelial cell damage.
Background Host factors such as angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease, serine-subtype-2 (TMPRSS2) are important factors for SARS-CoV-2 infection. Clinical and pre-clinical studies demonstrated that RAAS-blocking agents can be safely used during a SARS-CoV-2 infection but it is unknown if DPP-4 inhibitors or SGLT2-blockers may promote COVID-19 by increasing the host viral entry enzymes ACE2 and TMPRSS2. Methods We investigated telmisartan, linagliptin and empagliflozin induced effects on renal and cardiac expression of ACE2, TMPRSS2 and key enzymes involved in RAAS (REN, AGTR2, AGT) under high-salt conditions in a non-diabetic experimental 5/6 nephrectomy (5/6 Nx) model. In the present study, the gene expression of Ace2, Tmprss2, Ren, Agtr2 and Agt was assessed with qRT-PCR and the protein expression of ACE2 and TMPRSS2 with immunohistochemistry in the following experimental groups: Sham + normal diet (ND) + placebo (PBO); 5/6Nx + ND + PBO; 5/6Nx + high salt-diet (HSD) + PBO; 5/6Nx + HSD + telmisartan; 5/6Nx + HSD + linagliptin; 5/6Nx + HSD + empagliflozin. Results In the kidney, the expression of Ace2 was not altered on mRNA level under disease and treatment conditions. The renal TMPRSS2 levels (mRNA and protein) were not affected, whereas the cardiac level was significantly increased in 5/6Nx rats. Intriguingly, the elevated TMPRSS2 protein expression in the heart was significantly normalized after treatment with telmisartan, linagliptin and empagliflozin. Conclusions Our study indicated that there is no upregulation regarding host factors potentially promoting SARS-CoV-2 virus entry into host cells when the SGLT2-blocker empagliflozin, telmisartan and the DPP4-inhibitor blocker linagliptin are used. The results obtained in a preclinical, experimental non-diabetic kidney failure model need confirmation in ongoing interventional clinical trials.
Objective:
We address two questions relevant to infants' exposure to potentially toxic arsenolipids, namely, are the arsenolipids naturally present in fish transported intact to a mother's milk, and what is the efficiency of this transport.
Methods:
We investigated the transport of arsenolipids and other arsenic species present in fish to mother's milk by analyzing the milk of a single nursing mother at 15 sampling times over a 3-day period after she had consumed a meal of salmon. Total arsenic values were obtained by elemental mass spectrometry, and arsenic species were measured by HPLC coupled to both elemental and molecular mass spectrometry.
Results:
Total arsenic increased from background levels (0.1 mu g As kg(-1)) to a peak value of 1.72 lig As kg(-1) eight hours after the fish meal. The pattern for arsenolipids was similar to that of total arsenic, increasing from undetectable background levels (< 0.01 mu g As kg(-1)) to a peak after eight hours of 0.45 mu g As kg(-1). Most of the remaining total arsenic in the milk was accounted for by arsenobetaine. The major arsenolipids in the salmon were arsenic hydrocarbons (AsHCs; 55 % of total arsenolipids), and these compounds were also the dominant arsenolipids in the milk where they contributed over 90 % of the total arsenolipids.
Conclusions:
Our study has shown that ca 2-3 % of arsenic hydrocarbons, natural constituents of fish, can be directly transferred unchanged to the milk of a nursing mother. In view of the potential neurotoxicity of AsHCs, the effects of these compounds on the brain developmental stage of infants need to be investigated.
Microplastics are a global environmental issue contaminating aquatic and terrestrial environments. They have been reported in atmospheric deposition, and indoor and outdoor air, raising concern for public health due to the potential for exposure. Moreover, the atmosphere presents a new vehicle for microplastics to enter the wider environment, yet our knowledge of the quantities, characteristics and pathways of airborne microplastics is sparse. Here we show microplastics in atmospheric deposition in a major population centre, central London. Microplastics were found in all samples, with deposition rates ranging from 575 to 1008 microplastics/m(2)/d. They were found in various shapes, of which fibrous microplastics accounted for the great majority (92%). Across all samples, 15 different petrochemical-based polymers were identified. Bivariate polar plots indicated dependency on wind, with different source areas for fibrous and non-fibrous airborne microplastics. This is the first evidence of airborne microplastics in London and confirms the need to include airborne pathways when consolidating microplastic impacts on the wider environment and human health.
Plant growth-promoting bacteria (PGPB) affect host physiological processes in various ways. This study aims at elucidating the dependence of bacterial-induced growth promotion on the plant genotype and characterizing plant metabolic adaptations to PGPB. Eighteen Arabidopsis thaliana accessions were inoculated with the PGPB strain Kosakonia radicincitans DSM 16656(T). Colonisation pattern was assessed by enhanced green fluorescent protein (eGFP)-tagged K. radicincitans in three A. thaliana accessions differing in their growth response. Metabolic impact of bacterial colonisation was determined for the best responding accession by profiling distinct classes of plant secondary metabolites and root exudates. Inoculation of 18 A. thaliana accessions resulted in a wide range of growth responses, from repression to enhancement. Testing the bacterial colonisation of three accessions did not reveal a differential pattern. Profiling of plant secondary metabolites showed a differential accumulation of glucosinolates, phenylpropanoids and carotenoids in roots. Analysis of root exudates demonstrated that primary and secondary metabolites were predominantly differentially depleted by bacterial inoculation. The plant genotype controls the bacterial growth promoting traits. Levels of lutein and beta-carotene were elevated in inoculated roots. Supplementing a bacterial suspension with beta-carotene increased bacterial growth, while this was not the case when lutein was applied, indicating that beta-carotene could be a positive regulator of plant growth promotion.
Insulin-Like Growth Factor Binding Protein 2 (IGFBP-2) and the Risk of Developing Type 2 Diabetes
(2019)
Recent studies suggest that insulin-like growth factor binding protein 2 (IGFBP-2) may protect against type 2 diabetes, but population-based human studies are scarce. We aimed to investigate the prospective association of circulating IGFBP-2 concentrations and of differential methylation in the IGFBP-2 gene with type 2 diabetes risk.
Aims/hypothesis This study aimed to evaluate associations of height as well as components of height (sitting height and leg length) with risk of type 2 diabetes and to explore to what extent associations are explainable by liver fat and cardiometabolic risk markers. Methods A case-cohort study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study comprising 26,437 participants who provided blood samples was designed. We randomly selected a subcohort of 2500 individuals (2029 diabetes-free at baseline and with anamnestic, anthropometrical and metabolic data for analysis). Of the 820 incident diabetes cases identified in the full cohort during 7 years of follow-up, 698 remained for analyses after similar exclusions. Results After adjustment for age, potential lifestyle confounders, education and waist circumference, greater height was related to lower diabetes risk (HR per 10 cm, men 0.59 [95% CI 0.47, 0.75] and women 0.67 [0.51, 0.88], respectively). Leg length was related to lower risk among men and women, but only among men if adjusted for total height. Adjustment for liver fat and triacylglycerols, adiponectin and C-reactive protein substantially attenuated associations between height and diabetes risk, particularly among women. Conclusions/interpretation We observed inverse associations between height and risk of type 2 diabetes, which was largely related to leg length among men. The inverse associations may be partly driven by lower liver fat content and a more favourable cardiometabolic profile.
Metabolic alterations precede cardiometabolic disease onset. Here we present ceramide- and dihydroceramide-profiling data from a nested case-cohort (type 2 diabetes [T2D, n = 775]; cardiovascular disease [CVD, n = 551]; random subcohort [n = 1137]) in the prospective EPIC-Potsdam study. We apply the novel NetCoupler-algorithm to link a data-driven (dihydro)ceramide network to T2D and CVD risk. Controlling for confounding by other (dihydro)ceramides, ceramides C18:0 and C22:0 and dihydroceramides C20:0 and C22:2 are associated with higher and ceramide C20:0 and dihydroceramide C26:1 with lower T2D risk. Ceramide C16:0 and dihydroceramide C22:2 are associated with higher CVD risk. Genome-wide association studies and Mendelian randomization analyses support a role of ceramide C22:0 in T2D etiology. Our results also suggest that (dh)ceramides partly mediate the putative adverse effect of high red meat consumption and benefits of coffee consumption on T2D risk. Thus, (dihydro)ceramides may play a critical role in linking genetic predisposition and dietary habits to cardiometabolic disease risk.
Housing in metabolic cages can induce a pronounced stress response. Metabolic cage systems imply housing mice on metal wire mesh for the collection of urine and feces in addition to monitoring food and water intake. Moreover, mice are single-housed, and no nesting, bedding, or enrichment material is provided, which is often argued to have a not negligible impact on animal welfare due to cold stress. We therefore attempted to reduce stress during metabolic cage housing for mice by comparing an innovative metabolic cage (IMC) with a commercially available metabolic cage from Tecniplast GmbH (TMC) and a control cage. Substantial refinement measures were incorporated into the IMC cage design. In the frame of a multifactorial approach for severity assessment, parameters such as body weight, body composition, food intake, cage and body surface temperature (thermal imaging), mRNA expression of uncoupling protein 1 (Ucp1) in brown adipose tissue (BAT), fur score, and fecal corticosterone metabolites (CMs) were included. Female and male C57BL/6J mice were single-housed for 24 h in either conventional Macrolon cages (control), IMC, or TMC for two sessions. Body weight decreased less in the IMC (females—1st restraint: −6.94%; 2nd restraint: −6.89%; males—1st restraint: −8.08%; 2nd restraint: −5.82%) compared to the TMC (females—1st restraint: −13.2%; 2nd restraint: −15.0%; males—1st restraint: −13.1%; 2nd restraint: −14.9%) and the IMC possessed a higher cage temperature (females—1st restraint: 23.7 °C; 2nd restraint: 23.5 °C; males—1st restraint: 23.3 °C; 2nd restraint: 23.5 °C) compared with the TMC (females—1st restraint: 22.4 °C; 2nd restraint: 22.5 °C; males—1st restraint: 22.6 °C; 2nd restraint: 22.4 °C). The concentration of fecal corticosterone metabolites in the TMC (females—1st restraint: 1376 ng/g dry weight (DW); 2nd restraint: 2098 ng/g DW; males—1st restraint: 1030 ng/g DW; 2nd restraint: 1163 ng/g DW) was higher compared to control cage housing (females—1st restraint: 640 ng/g DW; 2nd restraint: 941 ng/g DW; males—1st restraint: 504 ng/g DW; 2nd restraint: 537 ng/g DW). Our results show the stress potential induced by metabolic cage restraint that is markedly influenced by the lower housing temperature. The IMC represents a first attempt to target cold stress reduction during metabolic cage application thereby producing more animal welfare friendlydata.
Background: Being an essential trace element, copper is involved in diverse physiological processes. However, excess levels might lead to adverse effects. Disrupted copper homeostasis, particularly in the brain, has been associated with human diseases including the neurodegenerative disorders Wilson and Alzheimer?s disease. In this context, astrocytes play an important role in the regulation of the copper homeostasis in the brain and likely in the prevention against neuronal toxicity, consequently pointing them out as a potential target for the neurotoxicity of copper. Major toxic mechanisms are discussed to be directed against mitochondria probably via oxidative stress. However, the toxic potential and mode of action of copper in astrocytes is poorly understood, so far. Methods: In this study, excess copper levels affecting human astrocytic cell model and their involvement in the neurotoxic mode of action of copper, as well as, effects on the homeostasis of other trace elements (Mn, Fe, Ca and Mg) were investigated. Results: Copper induced substantial cytotoxic effects in the human astrocytic cell line following 48 h incubation (EC30: 250 ?M) and affected mitochondrial function, as observed via reduction of mitochondrial membrane potential and increased ROS production, likely originating from mitochondria. Moreover, cellular GSH metabolism was altered as well. Interestingly, not only cellular copper levels were affected, but also the homeostasis of other elements (Ca, Fe and Mn) were disrupted. Conclusion: One potential toxic mode of action of copper seems to be effects on the mitochondria along with induction of oxidative stress in the human astrocytic cell model. Moreover, excess copper levels seem to interact with the homeostasis of other essential elements such as Ca, Fe and Mn. Disrupted element homeostasis might also contribute to the induction of oxidative stress, likely involved in the onset and progression of neurodegenerative disorders. These insights in the toxic mechanisms will help to develop ideas and approaches for therapeutic strategies against copper-mediated diseases.
As an essential trace element, copper plays a pivotal role in physiological body functions. In fact, dysregulated copper homeostasis has been clearly linked to neurological disorders including Wilson and Alzheimer’s disease. Such neurodegenerative diseases are associated with progressive loss of neurons and thus impaired brain functions. However, the underlying mechanisms are not fully understood. Characterization of the element species and their subcellular localization is of great importance to uncover cellular mechanisms. Recent research activities focus on the question of how copper contributes to the pathological findings. Cellular bioimaging of copper is an essential key to accomplish this objective. Besides information on the spatial distribution and chemical properties of copper, other essential trace elements can be localized in parallel. Highly sensitive and high spatial resolution techniques such as LA-ICP-MS, TEM-EDS, S-XRF and NanoSIMS are required for elemental mapping on subcellular level. This review summarizes state-of-the-art techniques in the field of bioimaging. Their strengths and limitations will be discussed with particular focus on potential applications for the elucidation of copper-related diseases. Based on such investigations, further information on cellular processes and mechanisms can be derived under physiological and pathological conditions. Bioimaging studies might enable the clarification of the role of copper in the context of neurodegenerative diseases and provide an important basis to develop therapeutic strategies for reduction or even prevention of copper-related disorders and their pathological consequences.
Arsenolipids are lipid-soluble organoarsenic compounds, mainly occurring in marine organisms, with arsenic-containing hydrocarbons (AsHCs) and arsenic-containing fatty acids (AsFAs) representing two major subgroups. Recently, toxicity studies of several arsenolipids showed a high cytotoxic potential of those arsenolipids in human liver and bladder cells. Furthermore, feeding studies with Drosophila melanogaster indicated an accumulation of arsenolipids in the fruit fly’s brain. In this study, the neurotoxic potential of three AsHCs, two AsFAs and three metabolites (dimethylarsinic acid, thio/oxo-dimethylarsenopropanoic acid) was investigated in comparison to the toxic reference arsenite (iAsIII) in fully differentiated human brain cells (LUHMES cells). Thereby, in the case of AsHCs both the cell number and cell viability were reduced in a low micromolar concentration range comparable to iAsIII, while AsFAs and the applied metabolites were less toxic. Mechanistic studies revealed that AsHCs reduced the mitochondrial membrane potential, whereas neither iAsIII nor AsFAs had an impact. Furthermore, neurotoxic mechanisms were investigated by examining the neuronal network. Here, AsHCs massively disturbed the neuronal network and induced apoptotic effects, while iAsIII and AsFAs showed comparatively lesser effects. Taking into account the substantial in vitro neurotoxic potential of the AsHCs and the fact that they could transfer across the physiological barriers of the brain, a neurotoxic potential in vivo for the AsHCs cannot be excluded and needs to be urgently characterized.
Scope: In the general population exposure to arsenic occurs mainly via diet. Highest arsenic concentrations are found in seafood, where arsenic is present predominantly in its organic forms including arsenolipids. Since recent studies have provided evidence that arsenolipids could reach the brain of an organism and exert toxicity in fully differentiated human neurons, this work aims to assess the neurodevelopmental toxicity of arsenolipids. Methods and results: Neurodevelopmental effects of three arsenic-containing hydrocarbons (AsHC), two arsenic-containing fatty acids (AsFA), arsenite and dimethylarsinic acid (DMA(V)) were characterized in pre-differentiated human neurons. AsHCs and arsenite caused substantial cytotoxicity in a similar, low concentration range, whereas AsFAs and DMA(V) were less toxic. AsHCs were highly accessible for cells and exerted pronounced neurodevelopmental effects, with neurite outgrowth and the mitochondrial membrane potential being sensitive endpoints; arsenite did not substantially decrease those two endpoints. In fully differentiated neurons, arsenite and AsHCs caused neurite toxicity. Conclusion: These results indicate for a neurodevelopmental potential of AsHCs. Taken into account the possibility that AsHCs might easily reach the developing brain when exposed during early life, neurotoxicity and neurodevelopmental toxicity cannot be excluded. Further studies are needed in order to progress the urgently needed risk assessment.
Background
Biomarker-based analyses are commonly reported in observational epidemiological studies; however currently there are no specific study quality assessment tools to assist evaluation of conducted research. Accounting for study design and biomarker measurement would be important for deriving valid conclusions when conducting systematic data evaluation.
Methods
We developed a study quality assessment tool designed specifically to assess biomarker-based cross-sectional studies (BIOCROSS) and evaluated its inter-rater reliability. The tool includes 10-items covering 5 domains: ‘Study rational’, ‘Design/Methods’, ‘Data analysis’, ‘Data interpretation’ and ‘Biomarker measurement’, aiming to assess different quality features of biomarker cross-sectional studies. To evaluate the inter-rater reliability, 30 studies were distributed among 5 raters and intraclass correlation coefficients (ICC-s) were derived from respective ratings.
Results
The estimated overall ICC between the 5 raters was 0.57 (95% Confidence Interval (CI): 0.38–0.74) indicating a good inter-rater reliability. The ICC-s ranged from 0.11 (95% CI: 0.01–0.27) for the domain ‘Study rational’ to 0.56 (95% CI: 0.40–0.72) for the domain ‘Data interpretation’.
Conclusion
BIOCROSS is a new study quality assessment tool suitable for evaluation of reporting quality from cross-sectional epidemiological studies employing biomarker data. The tool proved to be reliable for use by biomedical scientists with diverse backgrounds and could facilitate comprehensive review of biomarker studies in human research.
Investigation of processes that contribute to the maintenance of genomic stability is one crucial factor in the attempt to understand mechanisms that facilitate ageing. The DNA damage response (DDR) and DNA repair mechanisms are crucial to safeguard the integrity of DNA and to prevent accumulation of persistent DNA damage. Among them, base excision repair (BER) plays a decisive role. BER is the major repair pathway for small oxidative base modifications and apurinic/apyrimidinic (AP) sites. We established a highly sensitive non-radioactive assay to measure BER incision activity in murine liver samples. Incision activity can be assessed towards the three DNA lesions 8-oxo-2’-deoxyguanosine (8-oxodG), 5-hydroxy-2’-deoxyuracil (5-OHdU), and an AP site analogue. We applied the established assay to murine livers of adult and old mice of both sexes. Furthermore, poly(ADP-ribosyl)ation (PARylation) was assessed, which is an important determinant in DDR and BER. Additionally, DNA damage levels were measured to examine the overall damage levels. No impact of ageing on the investigated endpoints in liver tissue were found. However, animal sex seems to be a significant impact factor, as evident by sex-dependent alterations in all endpoints investigated. Moreover, our results revealed interrelationships between the investigated endpoints indicative for the synergetic mode of action of the cellular DNA integrity maintaining machinery.
Background: Epidemiological studies suggest that an increased red meat intake is associated with a higher risk of type 2 diabetes, whereas an increased fiber intake is associated with a lower risk. Objectives: We conducted an intervention study to investigate the effects of these nutritional factors on glucose and lipid metabolism, body-fat distribution, and liver fat content in subjects at increased risk of type 2 diabetes. Methods: This prospective, randomized, and controlled dietary intervention study was performed over 6 mo. All groups decreased their daily caloric intake by 400 kcal. The "control" group (N = 40) only had this requirement. The "no red meat" group (N = 48) in addition aimed to avoid the intake of red meat, and the "fiber" group (N = 44) increased intake of fibers to 40 g/d. Anthropometric parameters and frequently sampled oral glucose tolerance tests were performed before and after intervention. Body-fat mass and distribution, liver fat, and liver iron content were assessed by MRI and single voxel proton magnetic resonance spectroscopy. Results: Participants in all groups lost weight (mean 3.3 +/- 0.5 kg, P < 0.0001). Glucose tolerance and insulin sensitivity improved (P < 0.001), and body and visceral fat mass decreased in all groups (P < 0.001). These changes did not differ between groups. Liver fat content decreased significantly (P < 0.001) with no differences between the groups. The decrease in liver fat correlated with the decrease in ferritin during intervention (r(2) = 0.08, P = 0.0021). This association was confirmed in an independent lifestyle intervention study (Tuebingen Lifestyle Intervention Program, N = 229, P = 0.0084). Conclusions: Our data indicate that caloric restriction leads to a marked improvement in glucose metabolism and body-fat composition, including liver-fat content. The marked reduction in liver fat might be mediated via changes in ferritin levels. In the context of caloric restriction, there seems to be no additional beneficial impact of reduced red meat intake and increased fiber intake on the improvement in cardiometabolic risk parameters. This trial was registered at clinicaltrials.gov as NCT03231839.
Dysfunctional islets of Langerhans are a hallmark of type 2 diabetes (T2D). We hypothesize that differences in islet gene expression alternative splicing which can contribute to altered protein function also participate in islet dysfunction. RNA sequencing (RNAseq) data from islets of obese diabetes-resistant and diabetes-susceptible mice were analyzed for alternative splicing and its putative genetic and epigenetic modulators. We focused on the expression levels of chromatin modifiers and SNPs in regulatory sequences. We identified alternative splicing events in islets of diabetes-susceptible mice amongst others in genes linked to insulin secretion, endocytosis or ubiquitin-mediated proteolysis pathways. The expression pattern of 54 histones and chromatin modifiers, which may modulate splicing, were markedly downregulated in islets of diabetic animals. Furthermore, diabetes-susceptible mice carry SNPs in RNA-binding protein motifs and in splice sites potentially responsible for alternative splicing events. They also exhibit a larger exon skipping rate, e.g., in the diabetes gene Abcc8, which might affect protein function. Expression of the neuronal splicing factor Srrm4 which mediates inclusion of microexons in mRNA transcripts was markedly lower in islets of diabetes-prone compared to diabetes-resistant mice, correlating with a preferential skipping of SRRM4 target exons. The repression of Srrm4 expression is presumably mediated via a higher expression of miR-326-3p and miR-3547-3p in islets of diabetic mice. Thus, our study suggests that an altered splicing pattern in islets of diabetes-susceptible mice may contribute to an elevated T2D risk.
Objective: Hormone secretion from metabolically active tissues, such as pancreatic islets, is governed by specific and highly regulated signaling pathways. Defects in insulin secretion are among the major causes of diabetes. The molecular mechanisms underlying regulated insulin secretion are, however, not yet completely understood. In this work, we studied the role of the GTPase ARFRP1 on insulin secretion from pancreatic 13-cells. <br /> Methods: A 13-cell-specific Arfrp1 knockout mouse was phenotypically characterized. Pulldown experiments and mass spectrometry analysis were employed to screen for new ARFRP1-interacting proteins. Co-immunoprecipitation assays as well as super-resolution microscopy were applied for validation. <br /> Results: The GTPase ARFRP1 interacts with the Golgi-associated PDZ and coiled-coil motif-containing protein (GOPC). Both proteins are co localized at the trans-Golgi network and regulate the first and second phase of insulin secretion by controlling the plasma membrane localization of the SNARE protein SNAP25. Downregulation of both GOPC and ARFRP1 in Min6 cells interferes with the plasma membrane localization of SNAP25 and enhances its degradation, thereby impairing glucose-stimulated insulin release from 13-cells. In turn, overexpression of SNAP25 as well as GOPC restores insulin secretion in islets from 13-cell-specific Arfrp1 knockout mice. <br /> Conclusion: Our results identify a hitherto unrecognized pathway required for insulin secretion at the level of trans-Golgi sorting. (c) 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The whey protein beta-lactoglobulin has been proposed as a transporter for covalent bound bioactive compounds in order to enhance their stability and reduce their sensory perception. The garlic derived compounds allicin and diallyl disulfide were bound covalently to the native and heat denatured protein. The binding site and the influence of the modification on the digestibility were determined by mass spectrometric analysis of the modified beta-lactoglobulin. Further, the conformation of the modified protein was assessed by circular dichroism and dynamic light scattering. The free thiol group of Cys(121) turned out to be the major binding site. After proteolysis with trypsin at pH 7 but not with pepsin at pH 2, a limited transfer to other cysteinyl residues was observed. The covalently bound ligands did not mask any proteolytic cleavage sites of pepsin, trypsin or chymotrypsin. The modified beta-lactoglobulin showed a native like conformation, besides a moderate loosening of protein folding. The covalent binding of organosulfur compounds to beta-lactoglobulin provides a bioactive ingredient without impairing the digestibility and functional properties of the protein. (C) 2015 Elsevier Ltd. All rights reserved.
Insulin is the main anabolic hormone secreted by 13-cells of the pancreas stimulating the assimilation and storage of glucose in muscle and fat cells. It modulates the postprandial balance of carbohydrates, lipids and proteins via enhancing lipogenesis, glycogen and protein synthesis and suppressing glucose generation and its release from the liver. Resistance to insulin is a severe metabolic disorder related to a diminished response of peripheral tissues to the insulin action and signaling. This leads to a disturbed glucose homeostasis that precedes the onset of type 2 diabetes (T2D), a disease reaching epidemic proportions. A large number of studies reported an association between elevated circulating fatty acids and the development of insulin resistance. The increased fatty acid lipid flux results in the accumulation of lipid droplets in a variety of tissues. However, lipid intermediates such as diacylglycerols and ceramides are also formed in response to elevated fatty acid levels. These bioactive lipids have been associated with the pathogenesis of insulin resistance. More recently, sphingosine 1-phosphate (S1P), another bioactive sphingolipid derivative, has also been shown to increase in T2D and obesity. Although many studies propose a protective role of S1P metabolism on insulin signaling in peripheral tissues, other studies suggest a causal role of S1P on insulin resistance. In this review, we critically summarize the current state of knowledge of S1P metabolism and its modulating role on insulin resistance. A particular emphasis is placed on S1P and insulin signaling in hepatocytes, skeletal muscle cells, adipocytes and pancreatic 13-cells. In particular, modulation of receptors and enzymes that regulate S1P metabolism can be considered as a new therapeutic option for the treatment of insulin resistance and T2D.
Sphingolipids are a class of lipids that share a sphingoid base backbone. They exert various effects in eukaryotes, ranging from structural roles in plasma membranes to cellular signaling. De novo sphingolipid synthesis takes place in the endoplasmic reticulum (ER), where the condensation of the activated C₁₆ fatty acid palmitoyl-CoA and the amino acid L-serine is catalyzed by serine palmitoyltransferase (SPT). The product, 3-ketosphinganine, is then converted into more complex sphingolipids by additional ER-bound enzymes, resulting in the formation of ceramides. Since sphingolipid homeostasis is crucial to numerous cellular functions, improved assessment of sphingolipid metabolism will be key to better understanding several human diseases. To date, no assay exists capable of monitoring de novo synthesis sphingolipid in its entirety. Here, we have established a cell-free assay utilizing rat liver microsomes containing all the enzymes necessary for bottom-up synthesis of ceramides. Following lipid extraction, we were able to track the different intermediates of the sphingolipid metabolism pathway, namely 3-ketosphinganine, sphinganine, dihydroceramide, and ceramide. This was achieved by chromatographic separation of sphingolipid metabolites followed by detection of their accurate mass and characteristic fragmentations through high-resolution mass spectrometry and tandem-mass spectrometry. We were able to distinguish, unequivocally, between de novo synthesized sphingolipids and intrinsic species, inevitably present in the microsome preparations, through the addition of stable isotope-labeled palmitate-d₃ and L-serine-d₃. To the best of our knowledge, this is the first demonstration of a method monitoring the entirety of ER-associated sphingolipid biosynthesis. Proof-of-concept data was provided by modulating the levels of supplied cofactors (e.g., NADPH) or the addition of specific enzyme inhibitors (e.g., fumonisin B₁). The presented microsomal assay may serve as a useful tool for monitoring alterations in sphingolipid de novo synthesis in cells or tissues. Additionally, our methodology may be used for metabolism studies of atypical substrates – naturally occurring or chemically tailored – as well as novel inhibitors of enzymes involved in sphingolipid de novo synthesis.
We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N-2-(1-MIM)-dG, N-6-(1-MIM)-dA] in six tissues, as well as protein adducts [tau N-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N-2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N-6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.
BACKGROUND: Recent studies indicate that the bioavailability of anthocyanins is extremely low. One of the possible reasons could be their binding to proteins. Therefore, the binding affinity of cyanidin-3-glucoside (Cy3glc) to HSA and alpha-amylase was investigated by the quenching of protein tryptophan fluorescence. From data obtained, the binding constants and the free Gibbs energy were calculated. The changes in conformation of the proteins tested were studied with circular dichroism and the influence of binding on alpha-amylase activity determined. RESULTS: Cy3glc quenched the tryptophan fluorescence and upon ligand binding a change in protein structure was observed related to the corresponding decrease in the et-amylase activity. The association constants of 25 to 77 x 10(3) L mol(-1) were calculated for different proteins, indicating weak interactions of non-covalent nature. Competitive binding with HSA in the presence of 8-anilino-1-naphthalene sulfonic acid suggest involvement of hydrophobic interactions, in the case of HSA the possible site being subdomain IIA. CONCLUSION: The strongest affinity of Cy3glc for HSA being at pH 7 underlines its potential in transport and distribution of the phenolic compounds in organisms. An influence on salivary amylase activity is possible when drinking berry juices with high anthocyanins content.
Scope: Flavan-3-ols are abundant polyphenols in human nutrition and are associated with beneficial health effects. The aim of this study was to comparatively investigate the metabolic fate of (-)-epicatechin, procyanidin B1, and polymeric procyanidins in a randomized cross-over study in humans.
Methods and results: Parent compounds, conjugates, and microbial metabolites were determined in plasma, urine, and faeces by HPLC-MS and GC-MS/MS. Glucuronidated, sulfated, and methylated (-)-epicatechin and 5-(3',4'-dihydroxyphenyl)-valerolactone were the dominant metabolites in blood and urine. In addition, minor amounts of procyanidin B1 and 4-hydroxy-5-(3',4'-dihydroxyphenyl) valeric acid and their conjugated metabolites were detected. The formation of 5-(3',4'-dihydroxyphenyl)-valerolactone and 4-hydroxy-5-(3',4'-dihydroxyphenyl) valeric acid varied largely between individuals as well as with the degree of polymerization of flavan-3-ols. Monomer units were not detectable in plasma or urine after procyanidin B1 and polymeric procyanidin intake. No correlation was found between the intake of flavan-3-ols and the occurrence of phenolic acids in blood and urine or the phenolic compound profiles in faeces.
Conclusion: In addition to conjugated metabolites derived from the absorption of monomeric flavan-3-ols, 5-(3',4' -dihydroxyphenyl)-valerolactone represents an important in vivo metabolite of (-)-epicatechin and procyanidin B1 produced by the gut microbiota.
Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting- incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.
A tight hormonal control of energy homeostasis is of pivotal relevance for animals. Recent evidence suggests an involvement of the nuclear receptor NR1i3 (CAR). Fasting induces CAR by largely unknown mechanisms and CAR-deficient mice are defective in fasting adaptation. In rat hepatocytes CAR was induced by WY14643, a PPARalpha-agonist. A DR1 motif in the CAR promoter was necessary and sufficient for this control. The PPARalpha-dependent increase in CAR potentiated the phenobarbital-induced transcription of the prototypical CAR-dependent gene CYP2B1. Since free fatty acids are natural ligands for PPARalpha, a fasting-induced increase in free fatty acids might induce CAR. In accordance with this hypothesis, CAR induction by fasting was abrogated in PPARalpha-deficient mice.
Sarcopenia
(2020)
Sarcopenia represents a muscle-wasting syndrome characterized by progressive and generalized degenerative loss of skeletal muscle mass, quality, and strength occurring during normal aging. Sarcopenia patients are mainly suffering from the loss in muscle strength and are faced with mobility disorders reducing their quality of life and are, therefore, at higher risk for morbidity (falls, bone fracture, metabolic diseases) and mortality. <br /> Several molecular mechanisms have been described as causes for sarcopenia that refer to very different levels of muscle physiology. These mechanisms cover e. g. function of hormones (e. g. IGF-1 and Insulin), muscle fiber composition and neuromuscular drive, myo-satellite cell potential to differentiate and proliferate, inflammatory pathways as well as intracellular mechanisms in the processes of proteostasis and mitochondrial function. <br /> In this review, we describe sarcopenia as a muscle-wasting syndrome distinct from other atrophic diseases and summarize the current view on molecular causes of sarcopenia development as well as open questions provoking further research efforts for establishing efficient lifestyle and therapeutic interventions.
Epigenetic DNA methylation of EBI3 modulates human interleukin-35 formation via NFkB signaling
(2021)
Ulcerative colitis (UC), a severe chronic disease with unclear etiology that is associated with increased risk for colorectal cancer, is accompanied by dysregulation of cytokines. Epstein-Barr virus-induced gene 3 (EBI3) encodes a subunit in the unique heterodimeric IL-12 cytokine family of either pro- or anti-inflammatory function. After having recently demonstrated that upregulation of EBI3 by histone acetylation alleviates disease symptoms in a dextran sulfate sodium (DSS)-treated mouse model of chronic colitis, we now aimed to examine a possible further epigenetic regulation of EBI3 by DNA methylation under inflammatory conditions. Treatment with the DNA methyltransferase inhibitor (DNMTi) decitabine (DAC) and TNF alpha led to synergistic upregulation of EBI3 in human colon epithelial cells (HCEC). Use of different signaling pathway inhibitors indicated NF kappa B signaling was necessary and proportional to the synergistic EBI3 induction. MALDI-TOF/MS and HPLC-ESIMS/MS analysis of DAC/TNF alpha-treated HCEC identified IL-12p35 as the most probable binding partner to form a functional protein. EBI3/IL-12p35 heterodimers (IL-35) induce their own gene upregulation, something that was indeed observed in HCEC cultured with media from previously DAC/TNF alpha-treated HCEC. These results suggest that under inflammatory and demethylating conditions the upregulation of EBI3 results in the formation of anti-inflammatory IL-35, which might be considered as a therapeutic target in colitis.
Intestinal release of dietary triglycerides via chylomicrons is the major contributor to elevated postprandial triglyceride levels. Dietary lipids can be transiently stored in cytosolic lipid droplets (LDs) located in intestinal enterocytes for later release. ADP ribosylation factor-related protein 1 (ARFRP1) participates in processes of LD growth in adipocytes and in lipidation of lipoproteins in liver and intestine. This study aims to explore the impact of ARFRP1 on LD organization and its interplay with chylomicron-mediated triglyceride release in intestinal-like Caco-2 cells. Suppression of Arfrp1 reduced release of intracellularly derived triglycerides (0.69-fold) and increased the abundance of transitional endoplasmic reticulum ATPase TERA/VCP, fatty acid synthase-associated factor 2 (FAF2) and perilipin 2 (Plin2) at the LD surface. Furthermore, TERA/VCP and FAF2 co-occurred more frequently with ATGL at LDs, suggesting a reduced adipocyte triglyceride lipase (ATGL)-mediated lipolysis. Accordingly, inhibition of lipolysis reduced lipid release from intracellular storage pools by the same magnitude as Arfrp1 depletion. Thus, the lack of Arfrp1 increases the abundance of lipolysis-modulating enzymes TERA/VCP, FAF2 and Plin2 at LDs, which might decrease lipolysis and reduce availability of fatty acids for triglyceride synthesis and their release via chylomicrons. (C) 2018 The Authors. Published by Elsevier Inc.
Objectives: Stroke, frequently a consequence of hypertension, is one of the leading causes of death and neurological disabilities worldwide. In the ischemic brain, levels of endothelin-1, one of the most potent vasoconstrictors, are raised. Anti-inflammatory and neuroprotective effects of endothelin antagonists after stroke have been described in literature. Based on these findings, we investigated the protective effect of the endothelin converting enzyme/neutral endopeptidase blocker, SLV 338, in salt-loaded, stroke-prone, spontaneously hypertensive rats.
Methods: Male, 8-week-old spontaneously hypertensive stroke-prone rats were put on a high salt diet and treated with either 30 mg/kg or 100 mg/kg SLV 338 or vehicle for 27 weeks. Blood pressure, neurological outcome, body weight, and mortality were investigated throughout treatment. In weeks 1 and 9, animals were housed in metabolic cages for collection of urinary and blood samples and assessment of salt water and food intake. In weeks 22 and 27, additional blood samples were taken. At the end of the study, all brains were analyzed using magnetic resonance imaging.
Results: SLV 338 was well tolerated in all animals. Neurological outcome and infarct size were similar in all groups. Albuminuria was considerably delayed and the incidence of stroke significantly lowered in treated animals. In spontaneously hypertensive stroke-prone rats, treatment with SLV 338 significantly (P=0.01) improved survival in comparison to the vehicle treated group in a blood pressure-independent manner.
Discussion: Our data in spontaneously hypertensive stroke-prone rats demonstrate that combined endothelin converting enzyme/neutral endopeptidase inhibition could offer a new therapeutic approach for primary stroke prevention and improvement of mortality. The mechanism seems to be blood pressure-independent.
Odd-chain fatty acids (OCFA) are inversely associated with type-2-diabetes in epidemiological studies. They are considered as a biomarker for dairy intake because fermentation in ruminants yields high amounts of propionate, which is used as the primer for lipogenesis. Recently, we demonstrated endogenous OCFA synthesis from propionate in humans and mice, but how this is affected by microbial colonization is still unexplored. Here, we investigated the effect of increasing microbiota complexity on hepatic lipid metabolism and OCFA levels in different dietary settings. Germ-free (GF), gnotobiotic (SIH, simplified human microbiota) or conventional (CONV) C3H/HeOuJ-mice were fed a CHOW or high-fat diet with inulin (HFI) to induce microbial fermentation. We found that hepatic lipogenesis was increased with increasing microbiota complexity, independently of diet. In contrast, OCFA formation was affected by diet as well as microbiota. On CHOW, hepatic OCFA and intestinal gluconeogenesis decreased with increasing microbiota complexity (GF > SIH > CONV), while cecal propionate showed a negative correlation with hepatic OCFA. On HFI, OCFA levels were highest in SIH and positively correlated with cecal propionate. The propionate content in the CHOW diet was 10 times higher than that of HFI. We conclude that bacterial propionate production affects hepatic OCFA formation, unless this effect is masked by dietary propionate intake.
Catechins and procyanidins are major polyphenols in plant-derived foods. Despite intensive studies in recent years, neither their biochemical nor their toxicological properties have been clarified sufficiently. This study aimed to compare the methylation of catechins and procyanidins by the enzyme catechol-O-methyltransferase (COMT) in vitro. We conducted incubations with rat liver cytosol and human placental cytosol including S-adenosyl-L-methionine. The set of substrates comprised the catechins (-)-epicatechin (EC) and (+)catechin (CAT), the procyanidin dimers B1, B2, B3, B4, B5, and B7 as well as procyanidin trimer C1. After extraction, metabolites were analyzed by means of liquid chromatography-electrospray ionizationmass spectrometry and liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. EC and CAT were converted to two monomethylated metabolites each by human and rat COMT, with the 3'-O-methyl derivatives being consistently the main metabolites. Furthermore, the flavanyl units of procyanidins were methylated consecutively, leading to monomethylated and dimethylated dimeric metabolites as well as monomethylated, dimethylated, and trimethylated C1 metabolites. The methylation status of each flavanyl unit was determined by means of mass spectrometric quinone-methide fragmentation patterns. In addition, molecular modeling studies were performed with the aim to predict the preferred site of methylation and to verify the experimental data. In conclusion, our results indicate that the degree and position of methylation depend clearly on the three-dimensional structure of the entire substrate molecule.
Background Neurofibromatosis type 1 (NF1) is a multi-organ disease caused by mutations in neurofibromin 1 (NF1). Amongst other features, NF1 patients frequently show reduced muscle mass and strength, impairing patients' mobility and increasing the risk of fall. The role of Nf1 in muscle and the cause for the NF1-associated myopathy are mostly unknown. Methods To dissect the function ofNf1in muscle, we created muscle-specific knockout mouse models for NF1, inactivatingNf1in the prenatal myogenic lineage either under the Lbx1 promoter or under the Myf5 promoter. Mice were analysed during prenatal and postnatal myogenesis and muscle growth. Results Nf1(Lbx1)and Nf1(Myf5)animals showed only mild defects in prenatal myogenesis. Nf1(Lbx1)animals were perinatally lethal, while Nf1(Myf5)animals survived only up to approximately 25 weeks. A comprehensive phenotypic characterization of Nf1(Myf5)animals showed decreased postnatal growth, reduced muscle size, and fast fibre atrophy. Proteome and transcriptome analyses of muscle tissue indicated decreased protein synthesis and increased proteasomal degradation, and decreased glycolytic and increased oxidative activity in muscle tissue. High-resolution respirometry confirmed enhanced oxidative metabolism in Nf1(Myf5)muscles, which was concomitant to a fibre type shift from type 2B to type 2A and type 1. Moreover, Nf1(Myf5)muscles showed hallmarks of decreased activation of mTORC1 and increased expression of atrogenes. Remarkably, loss of Nf1 promoted a robust activation of AMPK with a gene expression profile indicative of increased fatty acid catabolism. Additionally, we observed a strong induction of genes encoding catabolic cytokines in muscle Nf1(Myf5)animals, in line with a drastic reduction of white, but not brown adipose tissue. Conclusions Our results demonstrate a cell autonomous role for Nf1 in myogenic cells during postnatal muscle growth required for metabolic and proteostatic homeostasis. Furthermore, Nf1 deficiency in muscle drives cross-tissue communication and mobilization of lipid reserves.
Regular consumption of fruits and vegetables, which is related to high plasma levels of lipid-soluble micro-nutrients such as carotenoids and tocopherols, is linked to lower incidences of various age-related diseases. Differences in lipid-soluble micronutrient blood concentrations seem to be associated with age. Our retrospective analysis included men and women aged 22-37 and 60-85 years from the Berlin Aging Study II. Participants with simultaneously available plasma samples and dietary data were included (n = 1973). Differences between young and old groups were found for plasma lycopene, alpha-carotene, alpha-tocopherol, beta-cryptoxanthin (only in women), and gamma-tocopherol (only in men). beta-Carotene, retinol and lutein/zeaxanthin did not differ between young and old participants regardless of the sex. We found significant associations for lycopene, alpha-carotene (both inverse), alpha-tocopherol, gamma-tocopherol, and beta-carotene (all positive) with age. Adjusting for BMI, smoking status, season, cholesterol and dietary intake confirmed these associations, except for beta-carotene. These micronutrients are important antioxidants and associated with lower incidence of age-related diseases, therefore it is important to understand the underlying mechanisms in order to implement dietary strategies for the prevention of age-related diseases. To explain the lower lycopene and alpha-carotene concentration in older subjects, bioavailability studies in older participants are necessary.
Birth weight variation is influenced by fetal and maternal genetic and non-genetic factors, and has been reproducibly associated with future cardio-metabolic health outcomes. In expanded genome-wide association analyses of own birth weight (n = 321,223) and offspring birth weight (n = 230,069 mothers), we identified 190 independent association signals (129 of which are novel). We used structural equation modeling to decompose the contributions of direct fetal and indirect maternal genetic effects, then applied Mendelian randomization to illuminate causal pathways. For example, both indirect maternal and direct fetal genetic effects drive the observational relationship between lower birth weight and higher later blood pressure: maternal blood pressure-raising alleles reduce offspring birth weight, but only direct fetal effects of these alleles, once inherited, increase later offspring blood pressure. Using maternal birth weight-lowering genotypes to proxy for an adverse intrauterine environment provided no evidence that it causally raises offspring blood pressure, indicating that the inverse birth weight-blood pressure association is attributable to genetic effects, and not to intrauterine programming.
Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.
Hormonal regulation of neuronal mitochondrial unfolded protein response and its impact on metabolism
(2019)
The hypothalamus is the main brain area of central regulation of whole body metabolism through impacting food intake and energy expenditure. For the complex regulation, high amounts of energy are needed and mainly provided by mitochondria. Hence, mitochondrial function is crucial for cell homeostasis and modulates central insulin sensitivity. Thus, mitochondrial dysfunction is associated with insulin resistance in the brain and therefore is involved in the pathogenesis of type-2 diabetes (T2D). Mitochondrial health and protein homeostasis is propagated by mitochondrial stress responses like e.g. mitochondrial unfolded protein response (UPRmt). Therefore, studies regarding the regulation of mitochondrial homeostasis are crucial for understanding its effects on the central nervous system (CNS) for the progression of metabolic and nutrition-dependent disorders.
One main aim of this thesis was to investigate the metabolic regulation of mitochondrial stress responsiveness in the hypothalamus. The observed results showed that functional ERK-dependent insulin signaling is needed for regulation of mitochondrial stress response (MSR) genes and positively impacted the metabolism by controlling mitochondrial proteostasis without affecting mitochondrial biogenesis.
To further explore the role of MSR genes for brain cell homeostasis and its consequences for the metabolism, one of the key players - the mitochondrial chaperone heat shock protein 10 (Hsp10) – was studied in detail. Hsp10 expression was decreased in insulin-resistant, hyperglycemic db/db mice brains along with increased protein oxidation. Leptin, another key hormone in regulating metabolism, was able to induce Hsp10 in neurons. Appropriately, lentiviral-mediated knock down (KD) of Hsp10 introduced into hypothalamic CLU-183 cells induced mitochondrial dysfunction, altered mitochondrial dynamics and increased contact sites between mitochondria and endoplasmic reticulum (ER). In addition, Hsp10 KD caused cellular insulin resistance along with increasing oxidative stress specifically in mitochondrial fraction.
Interestingly, acute Hsp10 KD in the arcuate nucleus of the hypothalamus in C57BL/6N male mice did not change body weight or food intake, but it increased plasma leptin concentrations suggesting an effect on global leptin signaling. It increased hepatic markers of gluconeogenesis and hepatic insulin resistance along with features of low-grade inflammation.
Long-term studies of hypothalamic Hsp10 KD mice revealed unaltered systemic insulin sensitivity. The demonstrated increase in markers of hepatic gluconeogenesis of acute Hsp10 KD was still exhibited after 13 weeks, but insulin resistance in the liver was no longer observed.
In conclusion, hypothalamic insulin action regulates MSR and ensures proper mitochondrial function which positively affects metabolism. In addition, hypothalamic Hsp10 acts as a modulator of both insulin and leptin signaling and is identified as pivotal for the regulation of central mitochondrial function as well as insulin sensitivity in the brain and it impacts liver function. It may present a regulator of brain-liver crosstalk influencing hepatic gluconeogenesis and insulin sensitivity through a novel regulatory signaling mechanism.
Mutations in the gene encoding for filaggrin (FLG) are major predisposing factors for atopic dermatitis (AD). Besides genetic predisposition, immunological dysregulations considerably contribute to its pathophysiology. For example, thymic stromal lymphopoietin (TSLP) is highly expressed in lesional atopic skin and significantly contributes to the pathogenesis of AD by activating dendritic cells that then initiate downstream effects on, for example, T cells. However, little is known about the direct interplay between TSLP, filaggrin-deficient skin and other immune cells such as T lymphocytes. In the present study, FLG knockdown skin equivalents, characterised by intrinsically high TSLP levels, were exposed to activated CD4(+) T cells. T cell exposure resulted in an inflammatory phenotype of the skin equivalents. Furthermore, a distinct shift from a Th1/Th17 to a Th2/Th22 profile was observed following exposure of T cells to filaggrin-deficient skin equivalents. Interestingly, TSLP directly stimulated T cell migration exclusively in filaggrin-deficient skin equivalents even in the absence of dendritic cells, indicating a hitherto unknown role of TSLP in the pathogenesis of AD.
Observational studies from all over the world continue to find high prevalence rates of vitamin D insufficiency and deficiency in many populations, including pregnant women. Beyond its classical function as a regulator of calcium and phosphate metabolism, vitamin D elicits numerous effects in the human body. Current evidence highlights a vital role of vitamin D in mammalian gestation. During pregnancy, adaptations in maternal vitamin D metabolism lead to a physiologic increase of vitamin D levels, mainly because of an increased renal production, although other potential sources like the placenta are being discussed. A sufficient supply of mother and child with calcium and vitamin D during pregnancy ensures a healthy bone development of the fetus, whereas lack of either of these nutrients can lead to the development of rickets in the child. Moreover, vitamin D insufficiency during pregnancy has consistently been associated with adverse maternal and neonatal pregnancy outcomes. In multitudinous studies, low maternal vitamin D status was associated with a higher risk for pre-eclampsia, gestational diabetes mellitus and other gestational diseases. Likewise, several negative consequences for the fetus have been reported, including fetal growth restriction, increased risk of preterm birth and a changed susceptibility for later-life diseases. However, study results are diverging and causality has not been proven so far. Meta-analyses on the relationship between maternal vitamin D status and pregnancy outcomes revealed a wide heterogeneity of studied populations and the applied methodology in vitamin D assessment. Until today, clinical guidelines for supplementation cannot be based on high-quality evidence and it is not clear if the required intake for pregnant women differs from non-pregnant women. Long-term safety data of vitamin D supplementation in pregnant women has not been established and overdosing of vitamin D might have unfavorable effects, especially in mothers and newborns with mutations of genes involved in vitamin D metabolism. Reliable data from large observational and interventional randomized control trials are urgently needed as a basis for any detailed and safe recommendations for supplementation in the general population and, most importantly, in pregnant women. This is of utmost importance, as ensuring a sufficient vitamin D-supply of mother and child implies a great potential for the prevention of birth complications and development of diseases.
In older persons, the origin of malnutrition is often multifactorial with a multitude of factors involved. Presently, a common understanding about potential causes and their mode of action is lacking, and a consensus on the theoretical framework on the etiology of malnutrition does not exist. Within the European Knowledge Hub "Malnutrition in the Elderly (MaNuEL)," a model of "Determinants of Malnutrition in Aged Persons" (DoMAP) was developed in a multistage consensus process with live meetings and written feedback (modified Delphi process) by a multiprofessional group of 33 experts in geriatric nutrition. DoMAP consists of three triangle-shaped levels with malnutrition in the center, surrounded by the three principal conditions through which malnutrition develops in the innermost level: low intake, high requirements, and impaired nutrient bioavailability. The middle level consists of factors directly causing one of these conditions, and the outermost level contains factors indirectly causing one of the three conditions through the direct factors. The DoMAP model may contribute to a common understanding about the multitude of factors involved in the etiology of malnutrition, and about potential causative mechanisms. It may serve as basis for future research and may also be helpful in clinical routine to identify persons at increased risk of malnutrition.
Plant proteins have become increasingly important for ecological reasons. Rapeseed is a novel source of plant proteins with high biological value, but its metabolic impact in humans is largely unknown. A randomized, controlled intervention study including 20 healthy subjects was conducted in a crossover design. All participants received a test meal without additional protein or with 28 g of rapeseed protein isolate or soy protein isolate (control). Venous blood samples were collected over a 360-min period to analyze metabolites; satiety was assessed using a visual analog scale. Postprandial levels of lipids, urea, and amino acids increased following the intake of both protein isolates. The postprandial insulin response was lower after consumption of the rapeseed protein than after intake of the soy protein (p< 0.05), whereas the postmeal responses of glucose, lipids, interleukin-6, minerals, and urea were comparable between the two protein isolates. Interestingly, the rapeseed protein exerted stronger effects on postprandial satiety than the soy protein (p< 0.05). The postmeal metabolism following rapeseed protein intake is comparable with that of soy protein. The favorable effect of rapeseed protein on postprandial insulin and satiety makes it a valuable plant protein for human nutrition.
To explore the genetic determinants of obesity and Type 2 diabetes (T2D), the German Center for Diabetes Research (DZD) conducted crossbreedings of the obese and diabetes-prone New Zealand Obese mouse strain with four different lean strains (B6, DBA, C3H, 129P2) that vary in their susceptibility to develop T2D. Genome-wide linkage analyses localized more than 290 quantitative trait loci (QTL) for obesity, 190 QTL for diabetes-related traits and 100 QTL for plasma metabolites in the out-cross populations. A computational framework was developed that allowed to refine critical regions and to nominate a small number of candidate genes by integrating reciprocal haplotype mapping and transcriptome data. The efficiency of the complex procedure was demonstrated for one obesity QTL. The genomic interval of 35 Mb with 502 annotated candidate genes was narrowed down to six candidates. Accordingly, congenic mice retained the obesity phenotype owing to an interval that contains three of the six candidate genes. Among these the phospholipase PLA2G4A exhibited an elevated expression in adipose tissue of obese human subjects and is therefore a critical regulator of the obesity locus. Together, our broad and complex approach demonstrates that combined- and comparative-cross analysis exhibits improved mapping resolution and represents a valid tool for the identification of disease genes.
Aims: The nitric oxide and endothelin systems are key components of a local paracrine hormone network in the heart. We previously reported that diastolic dysfunction observed in mice lacking the endothelial nitric oxide synthase (eNOS-/-) can be prevented by a genetic overexpression of ET-1. Sexual dimorphisms have been reported in both ET-1 and NO systems. Particularly, eNOS-/- mice present sex related phenotypic differences.
Main methods: We used the ET-1 transgenic (ET+/+), eNOS-/-, and crossbred ET+/+ eNOS-/- mice, and wild type controls. We measured cardiac function by heart catheterization. Cardiac ventricles were collected for histological and molecular profiling.
Key findings: We report here that (i) the level of ET-1 expression in eNOS-/- mice was elevated in males but not in females. (ii) Left ventricular end-diastolic blood pressure was higher in male eNOS-/- mice than in females. (ii) eNOS-/- males but not females developed cardiomyocyte hypertrophy. (iv) Perivascular fibrosis of intra-cardiac arteries developed in female ET+/+ and eNOS-/- mice but not in males. Additionally, (v) the cardiac expression of metalloprotease-9 was higher in eNOS-/- males compared to females. Finally, (vi) cardiac proteome analysis revealed that the protein abundance of the oxidative stress related enzyme superoxide dismutase presented with sexual dimorphism in eNOS-/- and ET+/+ mice.
Significance: These results indicate that the cardiac phenotypes of ET-1 transgenic mice and eNOS knockout mice are sex specific. Since both systems are key players in the pathogenesis of cardiovascular diseases, our findings might be important in the context of gender differences in patients with such diseases. (C) 2013 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Background The cardiac nitric oxide and endothelin-1 (ET-1) systems are closely linked and play a critical role in cardiac physiology. The balance between both systems is often disturbed in cardiovascular diseases. To define the cardiac effect of excessive ET-1 in a status of nitric oxide deficiency, we compared left ventricular function and morphology in wild-type mice, ET-1 transgenic (ET+/+) mice, endothelial nitric oxide synthase knockout (eNOS(-/-)) mice, and ET(+/+)eNOS(-/-) mice.
Methods and results eNOS(-/-) and ET(+/+)eNOS(-/-) mice developed high blood pressure compared with wild-type and ET+/+ mice. Left ventricular catheterization showed that eNOS(-/-) mice, but not ET(+/+)eNOS(-/-), developed diastolic dysfunction characterized by increased end-diastolic pressure and relaxation constant tau. To elucidate the causal molecular mechanisms driving the rescue of diastolic function in ET(+/+)eNOS(-/-) mice, the cardiac proteome was analyzed. Two-dimensional gel electrophoresis coupled to mass spectrometry offers an appropriate hypothesis-free approach. ET-1 overexpression on an eNOS(-/-) background led to an elevated abundance and change in posttranslational state of antioxidant enzymes (e. g., peroxiredoxin-6, glutathione S-transferase mu 2, and heat shock protein beta 7). In contrast to ET(+/+)eNOS(-/-) mice, eNOS(-/-) mice showed an elevated abundance of proteins responsible for sarcomere disassembly (e. g., cofilin-1 and cofilin-2). In ET(+/+)eNOS(-/-) mice, glycolysis was favored at the expense of fatty acid oxidation.
Conclusion eNOS(-/-) mice developed diastolic dysfunction; this was rescued by ET-1 transgenic overexpression. This study furthermore suggests that cardiac ET-1 overexpression in case of eNOS deficiency causes specifically the regulation of proteins playing a role in oxidative stress, myocytes contractility, and energy metabolism.
Background: To assess the chronic effect of the DPP-4 inhibitor, linagliptin, alone, in combination with exenatide, and during exenatide withdrawal, in diet-induced obese (DIO) rats.
Methods: Female Wistar rats were exposed to a cafeteria diet to induce obesity. Animals were then dosed with vehicle or linagliptin (3 mg/kg PO) orally once-daily for a 28 day period. In a subsequent study, rats received exenatide (either 3 or 30 mu g/kg/day) or vehicle by osmotic mini-pump for 28 days. In addition, groups of animals were dosed orally with linagliptin either alone or in combination with a 3 mu g/kg/day exenatide dose for the study duration. In a final study, rats were administered exenatide (30 mu g/kg/day) or vehicle by osmotic mini-pump for eleven days. Subsequently, exenatide-treated animals were transferred to vehicle or continued exenatide infusion for a further ten days. Animals transferred from exenatide to vehicle were also dosed orally with either vehicle or linagliptin. In all studies, body weight, food and water intake were recorded daily and relevant plasma parameters and carcass composition were determined.
Results: In contrast to exenatide, linagliptin did not significantly reduce body weight or carcass fat in DIO rats versus controls. Linagliptin augmented the effect of exenatide to reduce body fat when given in combination but did not affect the body weight response. In rats withdrawn from exenatide, weight regain was observed such that body weight was not significantly different to controls. Linagliptin reduced weight regain after withdrawal of exenatide such that a significant difference from controls was evident.
Conclusions: These data demonstrate that linagliptin does not significantly alter body weight in either untreated or exenatide-treated DIO rats, although it delays weight gain after exenatide withdrawal. This finding may suggest the utility of DPP-4 inhibitors in reducing body weight during periods of weight gain.
The Caenorhabditis elegans (C. elegans) is a model organism that has been increasingly used in health and environmental toxicity assessments. The quantification of such elements in vivo can assist in studies that seek to relate the exposure concentration to possible biological effects.
Therefore, this study is the first to propose a method of quantitative analysis of 21 ions by ion chromatography (IC), which can be applied in different toxicity studies in C. elegans.
The developed method was validated for 12 anionic species (fluoride, acetate, chloride, nitrite, bromide, nitrate, sulfate, oxalate, molybdate, dichromate, phosphate, and perchlorate), and 9 cationic species (lithium, sodium, ammonium, thallium, potassium, magnesium, manganese, calcium, and barium).
The method did not present the presence of interfering species, with R2 varying between 0.9991 and 0.9999, with a linear range from 1 to 100 mu g L-1.
Limits of detection (LOD) and limits of quantification (LOQ) values ranged from 0.2319 mu g L-1 to 1.7160 mu g L-1 and 0.7028 mu g L-1 to 5.1999 mu g L-1, respectively.
The intraday and interday precision tests showed an Relative Standard Deviation (RSD) below 10.0 % and recovery ranging from 71.0 % to 118.0 % with a maximum RSD of 5.5 %.
The method was applied to real samples of C. elegans treated with 200 uM of thallium acetate solution, determining the uptake and bioaccumulated Tl+ content during acute exposure.
A novel common variant in DCST2 is associated with length in early life and height in adulthood
(2015)
Common genetic variants have been identified for adult height, but not much is known about the genetics of skeletal growth in early life. To identify common genetic variants that influence fetal skeletal growth, we meta-analyzed 22 genome-wide association studies (Stage 1; N = 28 459). We identified seven independent top single nucleotide polymorphisms (SNPs) (P < 1 x 10(-6)) for birth length, of which three were novel and four were in or near loci known to be associated with adult height (LCORL, PTCH1, GPR126 and HMGA2). The three novel SNPs were followed-up in nine replication studies (Stage 2; N = 11 995), with rs905938 in DC-STAMP domain containing 2 (DCST2) genome-wide significantly associated with birth length in a joint analysis (Stages 1 + 2; beta = 0.046, SE = 0.008, P = 2.46 x 10(-8), explained variance = 0.05%). Rs905938 was also associated with infant length (N = 28 228; P = 5.54 x 10(-4)) and adult height (N = 127 513; P = 1.45 x 10(-5)). DCST2 is a DC-STAMP-like protein family member and DC-STAMP is an osteoclast cell-fusion regulator. Polygenic scores based on 180 SNPs previously associated with human adult stature explained 0.13% of variance in birth length. The same SNPs explained 2.95% of the variance of infant length. Of the 180 known adult height loci, 11 were genome-wide significantly associated with infant length (SF3B4, LCORL, SPAG17, C6orf173, PTCH1, GDF5, ZNFX1, HHIP, ACAN, HLA locus and HMGA2). This study highlights that common variation in DCST2 influences variation in early growth and adult height.
This study aims to further mechanistically understand toxic modes of action after chronic inorganic arsenic exposure. Therefore long-term incubation studies in cultured cells were carried out, to display chronically attained changes, which cannot be observed in the generally applied in vitro short-term incubation studies. Particularly, the cytotoxic, genotoxic and epigenetic effects of an up to 21 days incubation of human urothelial (UROtsa) cells with pico- to nanomolar concentrations of iAs(III) and its metabolite thio-DMA(V) were compared. After 21 days of incubation, cytotoxic effects were strongly enhanced in the case of iAs(III) and might partly be due to glutathione depletion and genotoxic effects on the chromosomal level. These results are in strong contrast to cells exposed to thio-DMA(V). Thus, cells seemed to be able to adapt to this arsenical, as indicated among others by an increase in the cellular glutathione level. Most interestingly, picomolar concentrations of both iAs(III) and thio-DMA(V) caused global DNA hypomethylation in UROtsa cells, which was quantified in parallel by 5-medC immunostaining and a newly established, reliable, high resolution mass spectrometry (HRMS)-based test system. This is the first time that epigenetic effects are reported for thio-DMA(V); iAs(III) induced epigenetic effects occur in at least 8000 fold lower concentrations as reported in vitro before. The fact that both arsenicals cause DNA hypomethylation at really low, exposure-relevant concentrations in human urothelial cells suggests that this epigenetic effect might contribute to inorganic arsenic induced carcinogenicity, which for sure has to be further investigated in future studies.
Fifteen obese ponies were used in a body weight (BW) reduction programme (BWRP, daily energy intake: 7.0-8.4 MJ/100 kg BW). A frequently sampled intravenous glucose tolerance test was used to assess insulin sensitivity. Subcutaneous adipose tissue biopsies of the tail head were obtained for mRNA gene expression profiles of adiponectin, retinol-binding protein 4 (RBP4), interleukin 6 (IL-6) and macrophage activation marker (CD68) before and after BWRP. Blood samples were analysed for serum leptin, serum RBP4 and plasma adiponectin. Significant BW losses occurred with 7 MJ DE/100 kg BW. Serum leptin and RBP4 were initially similar between insulin-resistant (IR) and insulin-sensitive (IS) ponies, and both significantly decreased during BWRP. Compared with IS ponies, IR ponies initially had significantly lower plasma adiponectin levels. At the beginning of BWRP, mRNA expression of RBP4, adiponectin, IL-6 and CD68 was similar between IR and IS ponies. Plasma adiponectin was strongly related to IR, whereas serum leptin and RBP4 were closely linked to adiposity, independent of insulin sensitivity. Adipose tissue mRNA expression profiles did not clearly reflect these differences. However, the role of subcutaneous adipose tissue in IR remains open.
Identification and LC-MS/MS-based analyses of technical enzymes in wheat flour and baked products
(2016)
The use of technical enzymes in bakery industry is necessary for a consistent and good quality of baked products. Since the cultivation of cereals leads to low amounts of endogenous enzymes being present, a need of their commercial alternatives is becoming a routine process in order to meet the consumer quality demands. Targeted quantification proteomics-based methods are necessary for their detection to meet the regulatory criteria. Here, we initially report on the identification of Lipase FE-01, a lipase from fungus Thermomyces lanuginosus, as analyzed by SDS-PAGE, in-Gel digestion, and MALDI-TOF-MS. In further experiments, the focus of the study was directed toward an extensive use and optimization of in-solution enzymatic digestion in combination with LC-MS/MS techniques in identification of specific peptide markers and finally in utilization of the latter in delivering reproducible quantification data for several different technical enzymes (alpha-amylases, xylanase, and lipases from microbial origin) in complex matrices such as baked bread and wheat flour. Two digestion protocols (a fast option using thermocycler program and the well-established overnight method) were tested, and both of these can be successfully applied. The application of isotopically labeled analogs of the MRM targeted peptides as internal standards and the addition of an internal protein standard during the extraction/digestion experiment were compared to determine the optimal quantification algorithm of the recovered enzyme concentrations. Thus, a standardized sensitive LC-MS/MS method could be developed to determine technical enzymes as forthcoming ingredients in the prefabricated food formulations in concentrations lower than 10 ppm.
The application of technical enzymes is a potential tool in modulating the dough and baking quality of cereal products. No endogenous amylases (alpha- and beta-forms) are present in mature wheat grains; they may be synthesized or activated during germination. Hence, microbial alpha-amylases are added to the dough, being resistant to the endogenous alpha-amylase/trypsin inhibitors. Here, we report on the initial identification of two technical enzymes from a commercial sample based on an in-gel tryptic digestion coupled with MALDI-MS analysis. The primary component of the protein fraction with 51.3 kDa was alpha-amylase from Aspergillus species. A second major protein with 24.8 kDa was identified as endo-1,4-xylanase from Thermomyces lanuginosus. In the following experimental work up, a targeted proteomics approach utilizing the combination of specific proteolytic digestion of the added amylase and xylanase in wheat flour, dough or baked products, solid phase extraction of released peptides and their detection using LC-MS/MS was optimized. The targeted (MRM) MS/MS peptide signals showed that the peptide "ALSSALHER" (MW = 983) originating from amylase and "GWNPGLNAR" (MW = 983) from xylanase can be used to identify the corresponding technical enzymes added. Consequently, locally available baked products were tested and found to contain these enzymes as supplementary ingredients. (C) 2014 Elsevier Ltd. All rights reserved.
This study is focused on the synthesis and characterization of hydroxy-apo-10'-carotenal/quantum dot (QD) conjugates aiming at the in vivo visualization of beta-ionone, a carotenoid-derived volatile compound known for its important contribution to the flavor and aroma of many fruits, vegetables, and plants. The synthesis of nanoparticles bound to plant volatile precursors was achieved via coupling reaction of the QD to C-27-aldehyde which was prepared from alpha-ionone via 12 steps in 2.4% overall yield. The formation of the QD-conjugate was confirmed by measuring its fluorescence spectrum to observe the occurrence of fluorescence resonance energy transfer.
Background/Aims: To investigate the renal phenotype under conditions of an activated renal ET-1 system in the status of nitric oxide deficiency, we compared kidney function and morphology in wild-type, ET-1 transgenic (ET+/+), endothelial nitric oxide synthase knockout (eNOS-/-) and ET+/+eNOS-/- mice. Methods: We assessed blood pressure, parameters of renal morphology, plasma cystatin C, urinary protein excretion, expression of genes associated with glomerular filtration barrier and tissue remodeling, and plasma metabolites using metabolomics. Results: eNOS-/- and ET+/+eNOS-/- mice developed hypertension. Osteopontin, albumin and protein excretion were increased in eNOS-/- and restored in ET+/+eNOS-/- animals. All genetically modified mice developed renal interstitial fibrosis and glomerulosclerosis. Genes involved in tissue remodeling (serpinel, TIMP1, Collal, CCL2) were up-regulated in eNOS-/-, but not in ET+/+eNOS-/- mice. Plasma levels of free carnitine and acylcarnitines, amino acids, diacyl phosphatidylcholines, lysophosphatidylcholines and hexoses were descreased in eNOS-/- and were in the normal range in ET+/+eNOS-/- mice. Conclusion: eNOS-/- mice developed renal dysfunction, which was partially rescued by ET-1 overexpression in eNOS-/- mice. The metabolomics results suggest that ET-1 overexpression on top of eNOS knockout is associated with a functional recovery of mitochondria (rescue effect in 13-oxidation of fatty acids) and an increase in antioxidative properties (normalization of monounsaturated fatty acids levels). (C) 2015 The Author(s) Published by S. Karger AG, Basel
The determination of free 25-hydroxyvitamin D (25(OH)D) as compared to the analysis of total 25-hydroxyvitamin D might reflect better the vitamin D status during pregnancy, since vitamin D-binding protein (DBP) concentrations increase throughout pregnancy and the vast majority of 25(OH)D is tightly bound to DBP thus strongly influencing total 25(OH)D. The concentration of the biologically active free 25(OH)D - on the other hand - is much less dependent on the DBP concentrations. The study was conducted in May-June 2016 in 368 Caucasian pregnant healthy women - residents of Northeastern Germany. Free 25(OH)D was either measured directly by commercial ELISA kit or assessed by calculation via total 25(OH)D, DBP, and albumin serum concentrations. Regardless of the detection method, free 25(OH)D lowers in the 3rd trimester comparing to the 1st trimester (by 12% and 21%, p < 0.05 and p < 0.001, for measured and calculated free 25(OH)D, respectively), whereas total 25(OH)D was not decreased in late pregnancy. DBP rises with gestational age. Total 25(OH)D was not correlated with serum calcium (p = 0.251), whereas free 25(OH)D was significantly (p = 0.007 for measured free 25(OH)D and p < 0.001 for calculated free 25(OH)D) positively correlated with calcium. All 25(OH) D isoforms were significantly negatively correlated with bone-specific alkaline phosphatase (BSAP), however the correlation strength was the lowest with total 25(OH)D (rho = -0.108, p = 0.038), whereas both measured and calculated free 25(OH)D revealed better associations with BSAP (rho = -0.203 and rho = -0.211 for measured and calculated free 25(OH)D, respectively, p < 0.001 for both). We established pregnancy trimester specific reference intervals for free measured and calculated 25(OH)D and DBP. Both measured and calculated free 25(OH)D showed better correlations with parameters of the endocrine vitamin D system (calcium and BSAP). Both ways of measuring free 25(OH)D in pregnant women are suitable as novel laboratory parameter for vitamin D status monitoring during human pregnancy and might replace in the future the routine total 25(OH)D assessment.
Dipeptidyl peptidase (DPP)-4 inhibitors delay chronic kidney disease (CKD) progression in experimental diabetic nephropathy in a glucose-independent manner. Here we compared the effects of the DPP-4 inhibitor linagliptin versus telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. Animals were allocated to 1 of 4 groups: sham operated plus placebo; 5/6 nephrectomy plus placebo; 5/6 nephrectomy plus linagliptin; and 5/6 nephrectomy plus telmisartan. Interstitial fibrosis was significantly decreased by 48% with linagliptin but a non-significant 24% with telmisartan versus placebo. The urine albumin-to-creatinine ratio was significantly decreased by 66% with linagliptin and 92% with telmisartan versus placebo. Blood pressure was significantly lowered by telmisartan, but it was not affected by linagliptin. As shown by mass spectrometry, the number of altered peptide signals for linagliptin in plasma was 552 and 320 in the kidney. For telmisartan, there were 108 peptide changes in plasma and 363 in the kidney versus placebo. Linagliptin up-regulated peptides derived from collagen type I, apolipoprotein C1, and heterogeneous nuclear ribonucleoproteins A2/B1, a potential downstream target of atrial natriuretic peptide, whereas telmisartan up-regulated angiotensin II. A second study was conducted to confirm these findings in 5/6 nephrectomy wild-type and genetically deficient DPP-4 rats treated with linagliptin or placebo. Linagliptin therapy in wild-type rats was as effective as DPP-4 genetic deficiency in terms of albuminuria reduction. Thus, linagliptin showed comparable efficacy to telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. However, the underlying pathways seem to be different. Copyright (C) 2016, International Society of Nephrology. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
DNA-repair mechanisms enable cells to maintain their genetic information by protecting it from mutations that may cause malignant growth. Recent evidence suggests that specific DNA-repair enzymes contain ISCs (iron-sulfur clusters). The nuclear-encoded protein frataxin is essential for the mitochondrial biosynthesis of ISCs. Frataxin deficiency causes a neurodegenerative disorder named Friedreich's ataxia in humans. Various types of cancer occurring at young age are associated with this disease, and hence with frataxin deficiency. Mice carrying a hepatocyte- specific disruption of the frataxin gene develop multiple liver tumours for unresolved reasons. In the present study, we show that frataxin deficiency in murine liver is associated with increased basal levels of oxidative DNA base damage. Accordingly, eukaryotic V79 fibroblasts overexpressing human frataxin show decreased basal levels of these modifications, while prokaryotic Salmonella enterica serotype Typhimurium TA 104 strains transformed with human frataxin show decreased mutation rates. The repair rates of oxidative DNA base modifications in V79 cells overexpressing frataxin were significantly higher than in control cells. Lastly, cleavage activity related to the ISC-independent repair enzyme 8-oxoguanine glycosylase was found to be unaltered by frataxin overexpression. These findings indicate that frataxin modulates DNA-repair mechanisms probably due to its impact on ISC-dependent repair proteins, linking mitochondrial dysfunction to DNA repair and tumour initiation.
We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals
Friedreich's ataxia is an inherited neurodegenerative disease caused by the reduced expression of the mitochondrially active protein frataxin. We have previously shown that mice with a hepatocyte-specific frataxin knockout (AlbFxn(-/-)) develop multiple hepatic tumors in later life. In the present study, hepatic carbohydrate metabolism in AlbFxn(-/-) mice at an early and late life stage was analyzed. In young (5-week-old) AlbFxn(-/-) mice hepatic ATP, glucose-6-phosphate and glycogen levels were found to be reduced by similar to 74, 80 and 88%, respectively, when compared with control animals. This pronounced ATP, G6P and glycogen depletion in the livers of young mice reverted in older animals: while half of the mice die before 30 weeks of age, the other half reaches 17 months of age and exhibits glycogen, G6P and ATP levels similar to those in age-matched controls. A key event in this respect seems to be the up-regulation of GLUT1, the predominant glucose transporter in fetal liver parenchyma, which became evident in AlbFxn(-/-) mice being 5-12 weeks of age. The most significant histological findings in animals being 17 or 22 months of age were the appearance of multiple clear cell, mixed cell and basophilic foci throughout the liver parenchyma as well as the development of hepatocellular adenomas and carcinomas. The hepatocarcinogenic process in AlbFxn 2/2 mice shows remarkable differences regarding carbohydrate metabolism alterations when compared with all other chemically and virally driven liver cancer models described up to now.
Retinol-binding protein 4 (RBP4) has been suggested as new adipokine, possibly linking obesity to type 2 diabetes mellitus (T2DM). Since the kidneys are the main site of RBP4 degradation and since renal failure is a frequent co-morbid condition with diabetes mellitus, we evaluated the association among RBP4, renal function and T2DM in an Asian population. RBP4 serum levels were analyzed in 110 subjects (50 with T2DM) using an enzyme-linked immunosorbent assay (ELISA). Based on a cut-off estimated glomerular filtration rate (eGFR) of 60 ml/min per 1.73 m(2) (calculated according the abbreviated MDRD formula which uses serum creatinine level, age and gender) and on the T2DM status, subjects were assigned to four subgroups: Group A - controls with an eGFR > 60 ml/min per 1.73 m(2), Group B - controls with an eGFR < 60 ml/min per 1.73 m(2), Group C- T2DM subjects with an eGFR>60 ml/min per 1.73 m(2), and Group D - T2DM subjects with an eGFR <60 ml/ mm per 1.73 m(2). In both the T2DM and control groups, RBP4 levels were higher in subjects with an eGFR < 60 ml/min per 1.73 m(2) than in subjects with an eGFR >60 ml/min per 1.73 m(2). However, the difference was only significant between the control groups (p <0.05). After adjusting for age, gender, BMI, eGFR and the presence of T2DM, eGFR, not T2DM, was associated with plasma RBP4 levels (p<0.05). These results suggest among Asians the eGFR, but not the presence of T2DM, is a major determinant of RBP4 serum levels. The eGFR should be taken into account when evaluating the role of RBP4 in the pathogenesis of insulin resistance and T2DM.
Inhibition of p53 transactivation activity does not promote mutagen-induced transformation of IEC-18
(2004)
Nonoxidized, biologically active parathyroid hormone determines mortality in hemodialysis patients
(2013)
Background: It was shown that nonoxidized PTH (n-oxPTH) is bioactive, whereas the oxidation of PTH results in a loss of biological activity.
Methods: In this study we analyzed the association of n-oxPTH on mortality in hemodialysis patients using a recently developed assay system.
Results: Hemodialysis patients (224 men, 116 women) had a median age of 66 years. One hundred seventy patients (50%) died during the follow-up period of 5 years. Median n-oxPTH levels were higher in survivors (7.2 ng/L) compared with deceased patients (5.0 ng/L; P = .002). Survival analysis showed an increased survival in the highest n-oxPTH tertile compared with the lowest n-oxPTH tertile (chi(2), 14.3; P = 0008). Median survival was 1702 days in the highest n-oxPTH tertile, whereas it was only 453 days in the lowest n-oxPTH tertile. Multivariable-adjusted Cox regression showed that higher age increased odds for death, whereas higher n-oxPTH reduced the odds for death. Another model analyzing a subgroup of patients with intact PTH (iPTH) concentrations at baseline above the upper normal range of the iPTH assay (70 ng/L) revealed that mortality in this subgroup was associated with oxidized PTH but not with n-oxPTH levels.
Conclusions: The predictive power of n-oxPTH and iPTH on the mortality of hemodialysis patients differs substantially. Measurements of n-oxPTH may reflect the hormone status more precisely. The iPTH-associated mortality is most likely describing oxidative stress-related mortality.
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.
Seafood, including finfish, shellfish, and seaweed, is the largest contributor to arsenic (As) exposure in many human populations. In contrast to the predominance of inorganic As in water and many terrestrial foods, As in marine-derived foods is present primarily in the form of organic compounds. To date, human exposure and toxicological assessments have focused on inorganic As, while organic As has generally been considered to be nontoxic. However, the high concentrations of organic As in seafood, as well as the often complex As speciation, can lead to complications in assessing As exposure from diet. In this report, we evaluate the presence and distribution of organic As species in seafood, and combined with consumption data, address the current capabilities and needs for determining human exposure to these compounds. The analytical approaches and shortcomings for assessing these compounds are reviewed, with a focus on the best practices for characterization and quantitation. Metabolic pathways and toxicology of two important classes of organic arsenicals, arsenolipids and arsenosugars, are examined, as well as individual variability in absorption of these compounds. Although determining health outcomes or assessing a need for regulatory policies for organic As exposure is premature, the extensive consumption of seafood globally, along with the preliminary toxicological profiles of these compounds and their confounding effect on assessing exposure to inorganic As, suggests further investigations and process-level studies on organic As are needed to fill the current gaps in knowledge.
Fatty acid synthase (FASN) catalyzing the terminal steps in the de novo biogenesis of fatty acids is correlated with low survival and high disease recurrence in patients with bladder cancer. Pyruvate kinase M2 (PKM2) regulates the final step of glycolysis levels and provides a growth advantage to tumors. However, it is unclear whether the change of PKM2 has an effect on FASN and what is the mechanisms underlying. Here we describe a novel function of PKM2 in control of lipid metabolism by mediating transcriptional activation of FASN, showing the reduced expression of sterol regulatory element binding protein 1c (SREBP-1c). We first discovered that PKM2 physically interacts with the SREBP-1c using biochemical approaches, and downregulation of PKM2 reduced the expression of SREBP-1c by inactivating the AKT/mTOR signaling pathway, which in turn directly suppressed the transcription of major lipogenic genes FASN to reduce tumor growths. Furthermore, either PKM2 inhibitor-Shikonin or FASN inhibitor-TVB-3166 alone induced a strong antiproliferative and anticolony forming effect in bladder cancer cell line. The combination of both inhibitors exhibits a super synergistic effect on blocking the bladder cancer cells growth. It provides a new target and scientific basis for the treatment of bladder cancer.
Arsenic-containing lipids (arsenolipids) are natural products present in fish and algae. Because these compounds occur in foods, there is considerable interest in their human toxicology. We report the synthesis and characterization of seven arsenic-containing lipids, including six natural products. The compounds comprise dimethylarsinyl groups attached to saturated long-chain hydrocarbons (three compounds), saturated long-chain fatty acids (two compounds), and monounsaturated long chain fatty acids (two compounds). The arsenic group was introduced through sodium dimethylarsenide or bis(dimethylarsenic) oxide. The latter route provided higher and more reproducible yields, and consequently, this pathway was followed to synthesize six of the seven compounds. Mass spectral properties are described to assist in the identification of these compounds in natural samples. The pure synthesized arsenolipids will be used for in vitro experiments with human cells to test their uptake, biotransformation, and possible toxic effects.
The majority of cases of community-acquired pneumonia are caused by Streptococcus pneumoniae and most studies on pneumococcal host interaction are based on cell culture or animal experiments. Thus, little is known about infections in human lung tissue.
Cyclooxygenase-2 and its metabolites play an important regulatory role in lung inflammation. Therefore, we established a pneumococcal infection model on human lung tissue demonstrating mitogen-activated protein kinase (MAPK)-dependent induction of cyclooxygenase-2 and its related metabolites.
In addition to alveolar macrophages and the vascular endothelium, cyclooxygenase-2 was upregulated in alveolar type II but not type I epithelial cells, which was confirmed in lungs of patients suffering from acute pneumonia. Moreover, we demonstrated the expression profile of all four E prostanoid receptors at the mRNA level and showed functionality of the E prostanoid(4) receptor by cyclic adenosine monophosphate production. Additionally, in comparison to previous studies, cyclooxygenase-2/prostaglandin E-2 related pro- and anti-inflammatory mediator regulation was partly confirmed in human lung tissue after pneumococcal infection.
Overall, cell type-specific and MAPK-dependent cyclooxygenase-2 expression and prostaglandin E-2 formation in human lung tissue may play an important role in the early phase of pneumococcal infections.
Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARgamma, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARgamma-, and LXR- regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARgamma-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages
Sections of fecal cylinders were analyzed using fluorescence in situ hybridization targeting 180 bacterial groups. Samples were collected from three groups of women (N = 20 each) treated for bacterial vaginosis with ciprofloxacin + metronidazole. Group A only received the combined antibiotic regimen, whereas the A/Sb group received concomitant Saccharomyces boulardii CNCM I-745 treatment, and the A.Sb group received S. boulardii prophylaxis following the 14-day antibiotic course. The number of stool cylinders analyzed was 188 out of 228 in group A, 170 out of 228 in group A/Sb, and 172 out of 216 in group Ash. The colonic biomass was organized into a separate mucus layer with no bacteria, a 10-30 mu m broad unstirred transitional layer enriched with bacteria, and a patchy fermentative area that mixed digestive leftovers with bacteria. The antibiotics suppressed bacteria mainly in the fermentative area, whereas abundant bacterial clades retreated to the transitional mucus and survived. As a result, the total concentration of bacteria decreased only by one order. These effects were lasting, since the overall recovery of the microbial mass, bacterial diversity and concentrations were still below pre-antibiotic values 4 months after the end of antibiotic treatment. Sb-prophylaxis markedly reduced antibiotic effects and improved the recovery rates. Since the colon is a sophisticated bioreactor, the study indicated that the spatial anatomy of its biomass was crucial for its function. (C) 2015 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Background: The goal was to study lipid profiles (TG, TC, LDL, HDL), effects on serum leptin, and fat tissue adiponectin, and resistin as well as body weight effects of Shan He Jian Fei Granules (SHJFG) in rats on a high fat diet.
Methods: Rats were randomly divided into five groups: normal control group fed with normal fat diet, rats on high fat diet receiving low dosage, middle dosage, high dosage of Shan He Jian Fei Granules (SHJFG) as well as a high fat diet group receiving placebo. Rats were treated for 8 weeks. Body weight and naso-anal length of each rat were recorded and Lee's index was calculated. Serum TG, TC, LDL, HDL and leptin concentrations were analyzed. The gene expressions of adiponectin and resistin in adipose tissues were tested by RT-PCR.
Results: Compared to the high-fat diet group, body weights, Lee's indexes, weight of fat tissues and serum TG, TC, LDL and leptin of SHJFG groups significantly decreased (p<0.05), whereas mRNA expressions of adiponectin and resistin of SHJFG groups significantly increased (p<0.05).
Conclusions: SHJFG could significantly lower body weight and serum TG, TC, and LDL of obese rats. The effects of SHJFG in lowering leptin synthesis and raising mRNA expression of adiponectin and resistin in fat tissues may act as part of the mechanisms in lowering body weight of obese rats. Further studies are needed to demonstrate whether SHJFG may also reduce overall cardiovascular morbidity and mortality like other lipid lowering drugs.
A novel method based on liquid-liquid extraction with subsequent gas chromatography separation and mass spectrometric detection (GC-MS) for the quantification of organic carbonates in cell culture materials is presented. Method parameters including the choice of extraction solvent, of extraction method and of extraction time were optimised and the method was validated. The setup allowed for determination within a linear range of more than two orders of magnitude. The limits of detection (LODs) were between 0.0002 and 0.002 mmol/L and the repeatability precisions were in the range of 1.5-12.9%. It could be shown that no matrix effects were present and recovery rates between 98 and 104% were achieved. The methodology was applied to cell culture models incubated with commercial lithium ion battery (LIB) electrolytes to gain more insight into the potential toxic effects of these compounds. The stability of the organic carbonates in cell culture medium after incubation was studied. In a porcine model of the blood-cerebrospinal fluid (CSF) barrier, it could be shown that a transfer of organic carbonates into the brain facing compartment took place.
Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya(R)), which acts as a functional S1P(1) antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P(1) activation profile and a sustained S1P(1) internalization in cultures of S1P(1)-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional antagonism. In vivo, both compounds induced a profound lymphopenia in mice. Finally, these substances showed efficacy in the EAE model, where they reduced clinical symptoms of the disease, and, on the molecular level, they reduced the T-cell infiltration and several inflammatory mediators in the brain and spinal cord. In summary, these data suggest that S1P(1)-selective compounds may have an advantage over fingolimod and siponimod, not only in MS but also in other autoimmune diseases.
Expression of pyruvate kinase M2 in preneoplastic hepatic foci of N-nitrosomorpholine-treated rats
(1999)
Direct stimulation of soluble guanylate cyclase (sGC) is emerging as a potential new approach for the treatment of renal disorders. sGC catalyzes the formation of cyclic guanosine monophosphate (cGMP), deficiency of which is implicated in the pathogenesis of chronic kidney disease (CKD). Therefore, new classes of drugs sGC stimulators and activators are being investigated in preclinical models under conditions where nitric oxide is deficient. In preclinical models with different etiologies of CKD, the sGC stimulators BAY 41-2272, BAY 41-8543, BAY 60-4552, riociguat and vericiguat and the sGC activators cinaciguat, ataciguat, BI 703704 and GSK2181236A have shown consistently renoprotective effects. Clinical trials are required to confirm these findings in humans, and to ascertain whether these agents could provide a future alternative to guideline-recommended treatments.
Background. In horses and ponies numerous medical conditions are known to be linked with inflammation in different tissues, especially in the liver. Besides affecting other metabolic pathways such as the expression of certain interleukins (IL), inflammation is associated with stress of the endoplasmic reticulum (ER). In particular, ER stress leads to adaptive stress response and can be measured by several markers of inflammatory and stress signalling pathways, like nuclear factor kappa B (NF-kB). Objectives. To investigate lipopolysaccharide (LPS)-induced inflammatory reactions and their modulation in horses and ponies by feeding a polyphenol-rich supplement consisting of green tea and curcuma. Methods. In a cross-over study, 11 animals were allocated to either a placebo or a supplement group and supplemented with 10 g of a blend of green tea and curcuma extract (GCE) or a placebo (calcium carbonate) once daily. After 21 days of supplementation, all animals underwent a LPS challenge to induce moderate systemic inflammation. Blood samples and liver biopsies were taken at standardized time points: 24 hours before and 12 hours after LPS challenge. Inflammatory blood parameters such as serum amyloid A (SAA), haptoglobin and retinol binding protein 4 (RBP4) were measured in serum. Hepatic mRNA levels of selected markers of inflammation such as haptoglobin, tumor necrosis factor alpha (TNF-alpha), IL-1 beta, IL-6, cluster of differentiation 68 (CD68), fibroblast growth factor 21 (FGF-21), NF-kappa B, activating transcription factor 4 (ATF4) were quantified by RT-qPCR. In addition, liver biopsies were examined histologically for inflammatory alterations. Results. Blood markers of acute inflammatory response increased after LPS challenge. In the liver, the proinflammatory cytokine IL-1 beta showed significantly lower mRNA levels after LPS challenge in the supplemented group (P = 0.04) compared to the placebo group. Levels of the hepatic CD68 mRNA increased significantly in the placebo group (P = 0.04). There were no significant differences between supplemented and placebo groups concerning other markers of inflammation and markers of ER stress within the liver. The number of hepatic macrophages were not different after LPS challenge in both feeding groups. Conclusion. LPS was able to induce inflammation but seemed less suitable to induce ER stress in the horses and ponies. The polyphenol-rich supplement showed some potential to reduce inflammatory responses. Nevertheless, the supplementation did not exert an overall anti-inflammatory effect in horses and ponies.
Background: Sex-specific differences in factors associated with aging and lifespan, such as sarcopenia and disease development, are increasingly recognized. The study aims to assess sex-specific aspects of the association between vitamin D insufficiency and low lean mass as well as between vitamin D insufficiency and the frailty phenotype.
Methods: A total of 1102 participants (51% women) from the Berlin Aging Study II were included in this cross-sectional study. Vitamin D insufficiency was defined as a 25(OH)D level <50 nmol/L. Lean mass was assessed with dual-energy x-ray absorptiometry and corrected by body mass index. Low lean mass was defined according to the Foundations for the National Institutes of Health Sarcopenia Project criteria (appendicular lean mass/body mass index <0.789 in men and <0.512 in women) and frailty defined according to the Fried criteria.
Results: In a risk factor adjusted analysis, the association of vitamin D insufficiency was significantly influenced by sex (P for interaction < 0.001). Men with vitamin D insufficiency had 1.8 times higher odds of having low lean mass, with no association between vitamin D insufficiency and low lean mass in women. Participants with vitamin D insufficiency had 1.5 higher odds of being prefrail/frail with no significant effect modification by sex.
Conclusions: We found notable sex-specific differences in the association of vitamin D insufficiency with low lean mass but not of vitamin D insufficiency with frailty. Vitamin D might play a relevant role in the loss of lean mass in men but not women and might be a biological marker of an unfavorable aging process associated with early development of frailty regardless of sex.