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Background: Despite mechanistic studies linking retinol and RBP4 (retinol binding protein 4) to the pathogenesis of cardiovascular diseases (CVD) and type 2 diabetes (T2D), epidemiological evidence is still conflicting. We investigated whether conflicting results of previous studies may be explained by differences in the association of retinol and RBP4 with cardiometabolic risk across subgroups with distinct sex, hypertension state, liver, or kidney function. Methods: We used case-cohorts nested in the EPIC (European Prospective Investigation Into Cancer and Nutrition)-Potsdam cohort (N=27 548) comprising a random sample of participants (n=2500) and all physician-verified cases of incident CVD (n=508, median follow-up time 8.2 years) and T2D (n=820, median follow-up time 6.3 years). We estimated nonlinear and linear multivariable-adjusted associations between the biomarkers and cardiometabolic diseases by restricted cubic splines and Cox regression, respectively, testing potential interactions with hypertension, liver, and kidney function. Additionally, we performed 2-sample Mendelian Randomization analyses in publicly available data. Results: The association of retinol with cardiometabolic risk was modified by hypertension state ( P interaction CVDP interaction T2Dper SD [95% CI]: CVD, 0.71 [0.56–0.90]; T2D, 0.81 [0.70–0.94]) but with higher cardiometabolic risk in normotensive participants (CVD, 1.32 [1.06–1.64]; T2D, 1.15 [0.98–1.36]). Our analyses also indicated a significant interaction between RBP4 and hypertension on CVD risk ( P interaction=0.04). Regarding T2D risk, we observed a u-shaped association with RBP4 in women ( P nonlinearity=0.01, P effect=0.02) and no statistically significant association in men. The biomarkers’ interactions with liver or kidney function were not statistically significant. Hypertension state-specific associations for retinol concentrations with cardiovascular mortality risk were replicated in National Health and Nutrition Examination Survey III. Conclusions: Our findings suggest a hypertension-dependent relationship between plasma retinol and cardiometabolic risk and complex interactions of RBP4 with sex and hypertension on cardiometabolic risk.

Subsequent to the dietary uptake of nitrate/nitrite in combination with acetaldehyde/ethanol, combination effects resulting from the sustained endogenous exposure to nitrite and acetaldehyde may be expected. This may imply locoregional effects in the upper gastrointestinal tract as well as systemic effects, such as a potential influence on endogenous formation of N-nitroso compounds (NOC). Salivary concentrations of the individual components nitrate and nitrite and acetaldehyde are known to rise after ingestion, absorption and systemic distribution, thereby reflecting their respective plasma kinetics and parallel secretion through the salivary glands as well as the microbial/enzymatic metabolism in the oral cavity. Salivary excretion may also occur with certain drug molecules and food constituents and their metabolites. Therefore, putative combination effects in the oral cavity and the upper digestive tract may occur, but this has remained largely unexplored up to now. In this Guest Editorial, published evidence on exposure levels and biokinetics of nitrate/nitrite/NOx, NOC and acetaldehyde in the organism is reviewed and knowledge gaps concerning combination effects are identified. Research is suggested to be initiated to study the related unresolved issues. published

Supplemental Digital Content is available in the text., Background: Many heart diseases can result in reduced pumping capacity of the heart muscle. A mismatch between ATP demand and ATP production of cardiomyocytes is one of the possible causes. Assessment of the relation between myocardial ATP production (MVATP) and cardiac workload is important for better understanding disease development and choice of nutritional or pharmacologic treatment strategies. Because there is no method for measuring MVATP in vivo, the use of physiology-based metabolic models in conjunction with protein abundance data is an attractive approach. METHOD: We developed a comprehensive kinetic model of cardiac energy metabolism (CARDIOKIN1) that recapitulates numerous experimental findings on cardiac metabolism obtained with isolated cardiomyocytes, perfused animal hearts, and in vivo studies with humans. We used the model to assess the energy status of the left ventricle of healthy participants and patients with aortic stenosis and mitral valve insufficiency. Maximal enzyme activities were individually scaled by means of protein abundances in left ventricle tissue samples. The energy status of the left ventricle was quantified by the ATP consumption at rest (MVATP[rest]), at maximal workload (MVATP[max]), and by the myocardial ATP production reserve, representing the span between MVATP(rest) and MVATP(max). Results: Compared with controls, in both groups of patients, MVATP(rest) was increased and MVATP(max) was decreased, resulting in a decreased myocardial ATP production reserve, although all patients had preserved ejection fraction. The variance of the energetic status was high, ranging from decreased to normal values. In both patient groups, the energetic status was tightly associated with mechanic energy demand. A decrease of MVATP(max) was associated with a decrease of the cardiac output, indicating that cardiac functionality and energetic performance of the ventricle are closely coupled. Conclusions: Our analysis suggests that the ATP-producing capacity of the left ventricle of patients with valvular dysfunction is generally diminished and correlates positively with mechanical energy demand and cardiac output. However, large differences exist in the energetic state of the myocardium even in patients with similar clinical or image-based markers of hypertrophy and pump function. Registration: URL: https://www.clinicaltrials.gov; Unique identifiers: NCT03172338 and NCT04068740.

When the amount of reactive oxygen species produced by human metabolism cannot be balanced by antioxidants, this phenomenon is commonly referred to as oxidative stress. It is hypothesised that diets with high amounts of plant food products may have a beneficial impact on oxidative stress status. However, few studies have examined whether a vegan diet is associated with lower oxidative stress compared to an omnivorous diet. The present cross-sectional study aimed to compare the levels of five oxidative stress biomarkers in vegans and omnivores. Data of 36 vegans and 36 omnivores from Germany and of 21 vegans and 18 omnivores from Finland were analysed. HPLC coupled with mass spectrometry or fluorescence detection and ELISA methods were used to measure the oxidative stress biomarkers malondialdehyde (MDA), protein carbonyls and 3-nitrotyrosine in plasma and 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-iso-prostaglandin F2α (8-iso-PGF2α) in 24 h urine. Analyses of variance and covariance, considering potential confounders, were used. Vegans and omnivores showed no differences in MDA and protein carbonyl concentrations. In Finnish but not in German vegans, the concentrations of 3-nitrotyrosine were lower compared to those in omnivores (

Persons with Down syndrome (DS) undergo a premature ageing with early onset of age-related diseases. The main endpoint of this study was the identification of blood circulating microRNAs (c-miRs) signatures characterizing DS ageing process. A discovery phase based on array was performed in plasma samples obtained from 3 young (31±2 years-old) and 3 elderly DS persons (66±2 years-old). Then, a validation phase was carried out for relevant miRs by RT-qPCR in an enlarged cohort of 43 DS individuals (from 19 up to 68 years-old). A group of 30 non-trisomic subjects, as representative of physiological ageing, was compared. In particular miR-628-5p, miR-152-3p, miR-28-5p, and let-7d-5p showed a lower level in younger DS persons (age ≤ 50 years) respect to the age-matched controls. Among those, miR-28-5p and let-7d-5p were found significantly decreased in physiological ageing ( oldest group ), thus they emerged as possible biomarkers of premature ageing in DS. Moreover, measuring blood levels of beta amyloid peptides, Aβ-42 was assessed at the lowest levels in physiological ageing and correlated with miR-28-5p and let-7d-5p in DS, while Aβ-40 correlated with miR-628-5p in the same cohort. New perspectives in terms of biomarkers are discussed.

Effects of feeding male rats during spermatogenesis a high-fat, high-sucrose and high-salt diet (HFSSD) over two generations (F0 and F1) on renal outcomes are unknown. Male F0 and F1 rats were fed either control diet (F0CD+F1CD) or HFSSD (F0HD+F1HD). The outcomes were glomerular filtration rate and urinary albumin excretion in F1 and F2 offspring. If both outcomes were altered a morphological and molecular assessment was done. F2 offspring of both sexes had a decreased GFR. However, increased urinary albumin excretion was only observed in female F2 F0HD+F1HD offspring compared with controls. F0HD+F1HD female F2 offspring developed glomerulosclerosis (+31%; p < .01) and increased renal interstitial fibrosis (+52%; p < .05). RNA sequencing followed by qRT-PCR validation showed that four genes (Enpp6, Tmem144, Cd300lf, and Actr3b) were differentially regulated in the kidneys of female F2 offspring. lncRNA XR-146683.1 expression decreased in female F0HD+F1HD F2 offspring and its expression was (r = 0.44, p = .027) correlated with the expression of Tmem144. Methylation of CpG islands in the promoter region of the Cd300lf gene was increased (p = .001) in female F2 F0HD+F1HD offspring compared to controls. Promoter CpG island methylation rate of Cd300lf was inversely correlated with Cd300lf mRNA expression in F2 female offspring (r = −0.483, p = .012). Cd300lf mRNA expression was inversely correlated with the urinary albumin-to-creatinine ratio in female F2 offspring (r = −0.588, p = .005). Paternal pre-conceptional unhealthy diet given for two generations predispose female F2 offspring to chronic kidney disease due to epigenetic alterations of renal gene expression. Particularly, Cd300lf gene promotor methylation was inversely associated with Cd300lf mRNA expression and Cd300lf mRNA expression itself was inversely associated with urinary albumin excretion in F2 female offspring whose fathers and grandfathers got a pre-conceptional unhealthy diet.

A decline of immune responses and dynamic modulation of the redox status are observed during aging and are influenced by trace elements such as copper, iodine, iron, manganese, selenium, and zinc. So far, analytical studies have focused mainly on single trace elements. Therefore, we aimed to characterize age-specific profiles of several trace elements simultaneously in serum and organs of adult and old mice. This allows for correlating multiple trace element levels and to identify potential patterns of age-dependent alterations. In serum, copper and iodine concentrations were increased and zinc concentration was decreased in old as compared to adult mice. In parallel, decreased copper and elevated iron concentrations were observed in liver. The age-related reduction of hepatic copper levels was associated with reduced expression of copper transporters, whereas the increased hepatic iron concentrations correlated positively with proinflammatory mediators and Nrf2-induced ferritin H levels. Interestingly, the age-dependent inverse regulation of copper and iron was unique for the liver and not observed in any other organ. The physiological importance of alterations in the iron/copper ratio for liver function and the aging process needs to be addressed in further studies.

BACKGROUND: A poor fat-soluble micronutrient (FMN) and a high oxidative stress status are associated with frailty. Our aim was to determine the cross-sectional association of FMNs and oxidative stress biomarkers [protein carbonyls (PrCarb) and 3-nitrotyrosine] with the frailty status in participants older than 65 years. METHODS: Plasma levels of vitamins A (retinol), D3 , E (alpha-tocopherol and gamma-tocopherol) and carotenoids (alpha-carotene and beta-carotene, lycopene, lutein/zeaxanthin, and beta-cryptoxanthin), PrCarb, and 3-nitrotyrosine were measured in 1450 individuals of the FRAILOMIC initiative. Participants were classified into robust, pre-frail, and frail using Fried's frailty criteria. Associations between biomarkers and frailty status were assessed by general linear and logistic regression models, both adjusted for cohort, season of blood sampling, gender, age, height, weight, and smoking. RESULTS: Robust participants had significantly higher vitamin D3 and lutein/zeaxanthin concentrations than pre-frail and frail subjects; had significantly higher gamma-tocopherol, alpha-carotene, beta-carotene, lycopene, and beta-cryptoxanthin concentrations than frail subjects, and had significantly lower PrCarb concentrations than frail participants in multivariate linear models. Frail subjects were more likely to be in the lowest than in the highest tertile for vitamin D3 (adjusted odds ratio: 2.15; 95% confidence interval: 1.42-3.26), alpha-tocopherol (2.12; 1.39-3.24), alpha-carotene (1.69; 1.00-2.88), beta-carotene (1.84; 1.13-2.99), lycopene (1.94; 1.24-3.05), lutein/zeaxanthin (3.60; 2.34-5.53), and beta-cryptoxanthin (3.02; 1.95-4.69) and were more likely to be in the highest than in the lowest tertile for PrCarb (2.86; 1.82-4.49) than robust subjects in multivariate regression models. CONCLUSIONS: Our study indicates that both low FMN and high PrCarb concentrations are associated with pre-frailty and frailty.

SCOPE Despite its beneficial properties, higher adiponectin concentrations are paradoxically associated with mortality in advanced age. Several mechanisms are being discussed. However, little is known about postprandial regulation of adiponectin in older adults. We assessed age-specific differences of the adiponectin response to different test meals considering potential determinants. METHODS AND RESULTS Older (n = 20) and younger (n = 22) women are randomized to a dextrose (DEX) or high-fat (HF) dietary challenge. Postprandial adiponectin and fibroblast growth factor 21 (FGF21) concentrations are measured before and 60, 120, 240 min after ingestion. We assessed postprandial changes and group differences using linear mixed models controlled for possible determinants. In younger women, postprandial adiponectin remains stable after both test meals. In contrast, adiponectin increases following DEX and decreases after HF in older women, irrespective of control variables. Postprandial adiponectin is positively associated with malondialdehyde and inversely associated with interleukin-6 following DEX and also negatively associated with metabolic parameters after both test meals. In older women, elevated postprandial FGF21 concentrations are associated with a higher adiponectin response (β = 30.7, 95% CI 10.6-50.8, p = 0.007). CONCLUSIONS Adiponectin response is associated with type of dietary challenge, age, and FGF21 response. Age-group differences are partly attributable to metabolic parameters and oxidative stress.

Dicarbonyl stress describes the increased formation of 1,2-dicarbonyl compounds and is associated with age-related pathologies. The role of dicarbonyl stress in healthy aging is poorly understood. In a preliminary study, we analyzed 1,2-dicarbonyl compounds, namely 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal (MGO) in plasma of older (25 months, n = 11) and younger (5 months, n = 14) male C57BL/6J (B6) mice via ultra performance liquid chromatography tandem mass spectrometry. Postprandial 3-DG was higher in younger compared to older mice, whereas no differences were found for GO and MGO. Subsequently, in the main study, we analyzed fasting serum of older women (OW, 72.4 ± 6.14 years, n = 19) and younger women (YW, 27.0 ± 4.42 years, n = 19) as well as older men (OM, 74.3 ± 5.20 years, n = 15) and younger men (YM, 27.0 ± 3.34, n = 15). Serum glucose, insulin, 1,2-dicarbonyl concentrations, and markers of oxidative stress were quantified. In a subgroup of this cohort, an oral dextrose challenge was performed, and postprandial response of 1,2-dicarbonyl compounds, glucose, and insulin were measured. In women, there were no age differences regarding fasting 1,2-dicarbonyl concentrations nor the response after the oral dextrose challenge. In men, fasting MGO was significantly higher in OM compared to YM (median: 231 vs 158 nM, p = .006), whereas no age differences in fasting 3-DG and GO concentrations were found. Glucose (310 ± 71.8 vs 70.8 ± 11.9 min·mmol/L) and insulin (7 149 ± 1 249 vs 2 827 ± 493 min·µIU/mL) response were higher in OM compared to YM, which did not translate into a higher 1,2-dicarbonyl response in older individuals. Overall, aging does not necessarily result in dicarbonyl stress, indicating that strategies to cope with 1,2-dicarbonyl formation can remain intact.

Ageing leaves characteristic traces in the DNA methylation make-up of the genome. However, the importance of DNA methylation in ageing remains unclear. The study of subtelomeric regions could give promising insights into this issue. Previously reported associations between susceptibility to age-related diseases and epigenetic instability at subtelomeres suggest that the DNA methylation profile of subtelomeres undergoes remodelling during ageing. In the present work, this hypothesis has been tested in the context of the European large-scale project MARK-AGE. In this cross-sectional study, we profiled the DNA methylation of chromosomes 5 and 21 subtelomeres, in more than 2000 age-stratified women and men recruited in eight European countries. The study included individuals from the general population as well as the offspring of nonagenarians and Down syndrome subjects, who served as putative models of delayed and accelerated ageing, respectively. Significant linear changes of subtelomeric DNA methylation with increasing age were detected in the general population, indicating that subtelomeric DNA methylation changes are typical signs of ageing. Data also show that, compared to the general population, the dynamics of age-related DNA methylation changes are attenuated in the offspring of centenarian, while they accelerate in Down syndrome individuals. This result suggests that subtelomeric DNA methylation changes reflect the rate of ageing progression. We next attempted to trace the age-related changes of subtelomeric methylation back to the influence of diverse variables associated with methylation variations in the population, including demographics, dietary/health habits and clinical parameters. Results indicate that the effects of age on subtelomeric DNA methylation are mostly independent of all other variables evaluated. Supplementary Information The online version contains supplementary material available at 10.1007/s11357-021-00347-9.

Liver fibrosis is a critical complication of obesity-induced fatty liver disease. Wnt1 inducible signaling pathway protein 1 (WISP1/CCN4), a novel adipokine associated with visceral obesity and insulin resistance, also contributes to lung and kidney fibrosis. The aim of the present study was to investigate the role of CCN4 in liver fibrosis in severe obesity. For this, human liver biopsies were collected from 35 severely obese humans (BMI 42.5 ± 0.7 kg/m2, age 46.7 ± 1.8 y, 25.7% males) during bariatric surgery and examined for the expression of CCN4, fibrosis, and inflammation markers. Hepatic stellate LX-2 cells were treated with human recombinant CCN4 alone or in combination with LPS or transforming growth factor beta (TGF-β) and examined for fibrosis and inflammation markers. CCN4 mRNA expression in the liver positively correlated with BMI and expression of fibrosis markers COL1A1, COL3A1, COL6A1, αSMA, TGFB1, extracellular matrix turnover enzymes TIMP1 and MMP9, and the inflammatory marker ITGAX/CD11c. In LX-2 cells, the exposure to recombinant CCN4 caused dose-dependent induction of MMP9 and MCP1. CCN4 potentiated the TGF-β-mediated induction of COL3A1, TIMP1, and MCP1 but showed no interaction with LPS treatment. Our results suggest a potential contribution of CCN4 to the early pathogenesis of obesity-associated liver fibrosis.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the “identify (OS) and treat (by low molecular weight antioxidants, LMWA)” approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different “antioxidants” are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the “identify and treat” approach is questionable.

Aging is a complex phenomenon that has detrimental effects on tissue homeostasis. The skeletal muscle is one of the earliest tissues to be affected and to manifest age-related changes such as functional impairment and the loss of mass. Common to these alterations and to most of tissues during aging is the disruption of the proteostasis network by detrimental changes in the ubiquitin-proteasomal system (UPS) and the autophagy-lysosomal system (ALS). In fact, during aging the accumulation of protein aggregates, a process mainly driven by increased levels of oxidative stress, has been observed, clearly demonstrating UPS and ALS dysregulation. Since the UPS and ALS are the two most important pathways for the removal of misfolded and aggregated proteins and also of damaged organelles, we provide here an overview on the current knowledge regarding the connection between the loss of proteostasis and skeletal muscle functional impairment and also how redox regulation can play a role during aging. Therefore, this review serves for a better understanding of skeletal muscle aging in regard to the loss of proteostasis and how redox regulation can impact its function and maintenance.

The ubiquitin-proteasomal-system (UPS) and the autophagy-lysosomal-system (ALS) are both highly susceptible for disturbances leading to the accumulation of cellular damage. A decline of protein degradation during aging results in the formation of oxidatively damaged and aggregated proteins finally resulting in failure of cellular functionality. Besides protein aggregation in response to oxidative damage, amyloids are a different type of protein aggregates able to distract proteostasis and interfere with cellular functionality. Amyloids are clearly linked to the pathogenesis of age-related degenerative diseases such as Alzheimer's disease. Human amylin is one of the peptides forming fibrils in β-sheet conformation finally leading to amyloid formation. In contrast to rodent amylin, human amylin is prone to form amyloidogenic aggregates, proposed to play a role in the pathogenesis of Type 2 Diabetes by impairing β-cell functionality. Since aggregates such as lipofuscin and β-amyloid are known to impair proteostasis, it is likely to assume similar effects for human amylin. In this review, we focus on the effects of IAPP on UPS and ALS and their role in amylin degradation, since both systems play a crucial role in maintaining proteome balance thereby influencing, at least in part, cellular fate and aging.

Frailty among elderly people leads to an increased risk for negative health outcomes. To prevent frailty, we need a better understanding of the underlying mechanisms and early detection of individuals at risk. Both may be served by identifying candidate (bio)markers, i.e. biomarkers and markers, for the physical, cognitive, and psychological frailty domains. We used univariate (Rank-ANOVA) and multivariate (elastic net) approaches on the RASIG study population (age range: 35–74 years, n = 2220) of the MARK-AGE study to study up to 331 (bio)markers between individuals with and without frailty for each domain. Biomarkers and markers identified by both approaches were studied further regarding their association with frailty using logistic regression. Univariately, we found lower levels of antioxidants, including β-cryptoxanthin and zeaxanthin, in those who were physically, cognitively or psychologically frail. Additionally, self-reported health was worse in these three frail groups. Multivariately, we observed lower levels of β-cryptoxanthin and zeaxanthin in the cognitively frail. Levels of these carotenoids were inversely associated with the risk of being cognitively frail after adjusting for confounders. Antioxidants and self-reported health are potential (bio)markers to detect persons at risk of becoming frail. The biomarkers identified may indicate the involvement of inflammation in frailty, especially for physical and cognitive frailty.

The proteasomal system is responsible for the turnover of damaged proteins. Because of its important functions in oncogenesis, inhibiting the proteasomal system is a promising therapeutic approach for cancer treatment. Bortezomib (BTZ) is the first proteasome inhibitor approved by FDA for clinical applications. However neuropathic side effects are dose limiting for BTZ as many other chemotherapeutic agents. Therefore second-generation proteasome inhibitors have been developed including carfilzomib (CFZ). Aim of the present work was investigating the mechanisms of peripheral neuropathy triggered by the proteasome inhibitor BTZ and comparing the pathways affected by BTZ and CFZ, respectively. Neural stem cells, isolated from the cortex of E14 mouse embryos, were treated with BTZ and CFZ and mass spectrometry was used to compare the global protein pool of treated cells. BTZ was shown to cause more severe cytoskeletal damage, which is crucial in neural cell integrity. Excessive protein carbonylation and actin filament destabilization were also detected following BTZ treatment that was lower following CFZ treatment. Our data on cytoskeletal proteins, chaperone system, and protein oxidation may explain the milder neurotoxic effects of CFZ in clinical applications.

4-Hydroxynonenal (HNE) is a major aldehydic product of lipid peroxidation known to exert several biological effects. Normal and malignant cells of the same origin express different sensitivity to HNE. We used human osteosarcoma cells (HOS) in different stages of differentiation in vitro, showing differences in mitosis, DNA synthesis, and alkaline phosphatase (ALP) staining. Differentiated HOS cells showed decreased proliferation (3H-thymidine incorporation), decreased viability (thiazolyl blue tetrazolium bromide-MTT), and increased apoptosis and necrosis (nuclear morphology by staining with 4′,6-diamidino-2-phenylindole-DAPI). Differentiated HOS also had less expressed c-MYC, but the same amount of c-FOS (immunocytochemistry). When exposed to HNE, differentiated HOS produced more reactive oxygen species (ROS) in comparison with undifferentiated HOS. To clarify this, we measured HNE metabolism by an HPLC method, total glutathione (GSH), oxidized GSH (ox GSH), glutathione transferase activity (GST), proteasomal activity by enzymatic methods, HNE-protein adducts by genuine ELISA and fatty acid composition by GC-MS in these cell cultures. Differentiated HOS cells had less GSH, lower HNE metabolism, increased formation of HNE-protein adducts, and lower proteasomal activity, in comparison to undifferentiated counterpart cells, while GST and oxGSH were the same. Fatty acids analyzed by GC-MS showed that there is an increase in C20:3 in differentiated HOS while the amount of C20:4 remained the same. The results showed that the cellular machinery responsible for protection against toxicity of HNE was less efficient in differentiated HOS cells. Moreover, differentiated HOS cells contained more C20:3 fatty acid, which might make them more sensitive to free radical-initiated oxidative chain reactions and more vulnerable to the effects of reactive aldehydes such as HNE. We propose that HNE might act as natural promotor of decay of malignant (osteosarcoma) cells in case of their differentiation associated with alteration of the lipid metabolism.

The analysis of copper (Cu) and zinc (Zn) along with their major serum carriers, albumin (Alb) and ceruloplasmin (Cp), could provide information on the capacity of humans to maintain homeostasis of metals (metallostasis). However, their relationship with aging, sex, body mass index, as well as with nutritional and inflammatory markers was never investigated in a large-scale study. Here, we report results from the European large-scale cross-sectional study MARK-AGE in which Cu, Zn, Alb, Cp, as well as nutritional and inflammatory parameters were determined in 2424 age-stratified participants (35–75 years), including the general population (RASIG), nonagenarian offspring (GO), a well-studied genetic model of longevity, and spouses of GO (SGO). In RASIG, Cu to Zn ratio and Cp to Alb ratio were higher in women than in men. Both ratios increased with aging because Cu and Cp increased and Alb and Zn decreased. Cu, Zn, Alb, and Cp were found associated with several inflammatory as well as nutritional biomarkers. GO showed higher Zn levels and higher Zn to Alb ratio compared to RASIG, but we did not observe significant differences with SGO, likely as a consequence of the low sample size of SGO and the shared environment. Our results show that aging, sex, body mass index, and GO status are characterized by different levels of Cu, Zn, and their serum carrier proteins. These data and their relationship with inflammatory biomarkers support the concept that loss of metallostasis is a characteristic of inflammaging.

Phenotype-specific omic expression patterns in people with frailty could provide invaluable insight into the underlying multi-systemic pathological processes and targets for intervention. Classical approaches to frailty have not considered the potential for different frailty phenotypes. We characterized associations between frailty (with/without disability) and sets of omic factors (genomic, proteomic, and metabolomic) plus markers measured in routine geriatric care. This study was a prevalent case control using stored biospecimens (urine, whole blood, cells, plasma, and serum) from 1522 individuals (identified as robust (R), pre-frail (P), or frail (F)] from the Toledo Study of Healthy Aging (R=178/P=184/F=109), 3 City Bordeaux (111/269/100), Aging Multidisciplinary Investigation (157/79/54) and InCHIANTI (106/98/77) cohorts. The analysis included over 35,000 omic and routine laboratory variables from robust and frail or pre-frail (with/without disability) individuals using a machine learning framework. We identified three protective biomarkers, vitamin D3 (OR: 0.81 [95% CI: 0.68-0.98]), lutein zeaxanthin (OR: 0.82 [95% CI: 0.70-0.97]), and miRNA125b-5p (OR: 0.73, [95% CI: 0.56-0.97]) and one risk biomarker, cardiac troponin T (OR: 1.25 [95% CI: 1.23-1.27]). Excluding individuals with a disability, one protective biomarker was identified, miR125b-5p (OR: 0.85, [95% CI: 0.81-0.88]). Three risks of frailty biomarkers were detected: pro-BNP (OR: 1.47 [95% CI: 1.27-1.7]), cardiac troponin T (OR: 1.29 [95% CI: 1.21-1.38]), and sRAGE (OR: 1.26 [95% CI: 1.01-1.57]). Three key frailty biomarkers demonstrated a statistical association with frailty (oxidative stress, vitamin D, and cardiovascular system) with relationship patterns differing depending on the presence or absence of a disability.

Torquetenovirus (TTV) viremia has been associated with increased mortality risk in the elderly population. This work aims to investigate TTV viremia as a potential biomarker of immunosenescence. We compared levels of circulating TTV in 1813 participants of the MARK-AGE project, including human models of delayed (offspring of centenarians [GO]) and premature (Down syndrome [DS]) immunosenescence. The TTV load was positively associated with age, cytomegalovirus (CMV) antibody levels, and the Cu/Zn ratio and negatively associated with platelets, total cholesterol, and total IgM. TTV viremia was highest in DS and lowest in GO, with intermediate levels in the SGO (spouses of GO) and RASIG (Randomly Recruited Age-Stratified Individuals From The General Population) populations. In the RASIG population, TTV DNA loads showed a slight negative association with CD3+T-cells and CD4+T-cells. Finally, males with ≥4log TTV copies/mL had a higher risk of having a CD4/CD8 ratio

Scope Several studies show that excessive lipid intake can cause hepatic steatosis. To investigate lipotoxicity on cellular level, palmitate (PA) is often used to highly increase lipid droplets (LDs). One way to remove LDs is autophagy, while it is controversially discussed if autophagy is also affected by PA. It is aimed to investigate whether PA-induced LD accumulation can impair autophagy and punicalagin, a natural autophagy inducer from pomegranate, can improve it. Methods and results To verify the role of autophagy in LD degradation, HepG2 cells are treated with PA and analyzed for LD and perilipin 2 content in presence of autophagy inducer Torin 1 and inhibitor 3-Methyladenine. PA alone seems to initially induce autophagy-related proteins but impairs autophagic-flux in a time-dependent manner, considering 6 and 24 h PA. To examine whether punicalagin can prevent autophagy impairment, cells are cotreated for 24 h with PA and punicalagin. Results show that punicalagin preserves expression of autophagy-related proteins and autophagic flux, while simultaneously decreasing LDs and perilipin 2. Conclusion Data provide new insights into the role of PA-induced excessive LD content on autophagy and suggest autophagy-inducing properties of punicalagin, indicating that punicalagin can be a health-beneficial compound for future research on lipotoxicity in liver.

Neurodegenerative diseases (NDD) such as Alzheimer's (AD) and Parkinson's disease (PD) are distinct clinical entities, however, the aggregation of key neuronal proteins, presumably leading to neuronal demise appears to represent a common mechanism. It has become evident, that advanced glycation end products (AGEs) trigger the accumulation of such modified proteins, which eventually contributes to pathological aspect of NDDs. Increased levels of AGEs are found in amyloid plaques in AD brains and in both advanced and early PD (incidental Lewy body disease). The molecular mechanisms by which AGE dependent modifications may modulate the susceptibility towards NDDs, however, remain enigmatic and it is unclear, whether AGEs may serve as biomarker of NDD. In the present study, we examined AGEs (CML: Carboxymethyllysine and CEL: Carboxyethyllysine), markers of oxidative stress and micronutrients in the plasma of PD and AD patients and controls. As compared to healthy controls, AD females displayed lower levels of CEL while higher levels of CML were found in AD and PD patients. A somewhat similar pattern was observed for protein carbonyls (PC), revealing lower values exclusively in AD females, whereas AD males displayed significantly higher values compared to healthy controls and PD. Sex-specific differences were also observed for other relevant markers such as malondialdehyde, 3-nitrotyrosine, γ -tocopherols, retinol, plasma proteins and α-carotene, while α-tocopherols, β-carotene, lutein/zeaxanthin, β-cryptoxanthin and lycopene showed no relevant association. Taken together, our study suggests yet unappreciated differences of the distribution of AGEs among the sexes in NDD. We therefore suggest to make a clear distinction between sexes when analyzing oxidative (AGEs)-related stress and carbonyl-related stress and vitamins., Graphical abstract Image 1, Highlights • As compared to healthy controls, AD females displayed lower levels of CEL (carboxyethyllysine) while higher levels of CML (carboxymethyllysine) were found in AD and PD patients. • A somewhat similar pattern was observed for protein carbonyls (PC), revealing lower values exclusively in AD females, whereas AD males displayed significantly higher values compared to healthy controls and PD. Presumed carbonlyated residues were identified the in the major proteins associated with neurodegenerative diseases. • Sex-specific differences were also observed for other relevant markers such as malondialdehyde, 3-nitrotyrosine, γ-tocopherols, retinol, plasma proteins and α-carotene, while α-tocopherols, β-carotene, lutein/zeaxanthin, β-cryptoxanthin and lycopene showed no relevant association.

Background and aims Non-alcoholic fatty liver disease (NAFLD) is becoming increasingly prevalent and nutrition intervention remains the most important therapeutic approach for NAFLD. Our aim was to investigate whether low- (LP) or high-protein (HP) diets are more effective in reducing liver fat and reversing NAFLD and which mechanisms are involved. Methods 19 participants with morbid obesity undergoing bariatric surgery were randomized into two hypocaloric (1500-1600 kcal/day) diet groups, a low protein (10E% protein) and a high protein (30E% protein), for three weeks prior to surgery. Intrahepatic lipid levels (IHL) and serum fibroblast growth factor 21 (FGF21) were measured before and after the dietary intervention. Autophagy flux, histology, mitochondrial activity, and gene expression analyses were performed in liver samples collected during surgery. Results IHL levels decreased by 42.6% in the HP group, but were not significantly changed in the LP group despite similar weight loss. Hepatic autophagy flux and serum FGF21 increased by 66.7% and 42.2%, respectively, after 3 weeks in the LP group only. Expression levels of fat uptake and lipid biosynthesis genes were lower in the HP group compared with those in the LP group. RNA-seq analysis revealed lower activity of inflammatory pathways upon HP diet. Hepatic mitochondrial activity and expression of β-oxidation genes did not increase in the HP group. Conclusions HP diet more effectively reduces hepatic fat than LP diet despite of lower autophagy and FGF21. Our data suggest that liver fat reduction upon HP diets result primarily from suppression of fat uptake and lipid biosynthesis.

Regular consumption of fruits and vegetables, which is related to high plasma levels of lipid-soluble micronutrients 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, α-carotene, α-tocopherol, β-cryptoxanthin (only in women), and γ-tocopherol (only in men). β-Carotene, retinol and lutein/zeaxanthin did not differ between young and old participants regardless of the sex. We found significant associations for lycopene, α-carotene (both inverse), α-tocopherol, γ-tocopherol, and β-carotene (all positive) with age. Adjusting for BMI, smoking status, season, cholesterol and dietary intake confirmed these associations, except for β-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 α-carotene concentration in older subjects, bioavailability studies in older participants are necessary., Graphical abstract Image 1, Highlights • Eating fruits and vegetables is linked to lower incidence of age-related diseases. • Different patterns of plasma antioxidant micronutrients in young vs. old subjects. • Lycopene, α-carotene, α-, and γ-tocopherol are associated with age. • Adjusting for BMI, smoking, season, cholesterol and diet confirmed associations. • Underlying mechanism could be altered bioavailability of micronutrients in aging.

Supplemental Digital Content is available in the text, Introduction: The mean age of the German population increased over the last years, which resulted in a higher prevalence of cardiovascular diseases, type 2 diabetes, cognitive impairment, sarcopenia and bone fractures. Current evidence indicates a preservation of human wellbeing in the elderly by a healthy diet, although the recommended macronutrient composition and quality remains unclear and needs further long-term investigation. In this context we investigate the effect of a specific dietary pattern on age-related disorders in a randomized controlled multi-center trial (RCT). Methods: We assess the effect of a specific dietary pattern (NutriAct) with a high proportion of unsaturated fat, plant proteins and fibres (fat 35%–40% of total energy (%E) of which 15%E–20%E monounsaturated fatty acids (MUFA) and 10%E–15%E polyunsaturated fatty acids (PUFA), 15%E–25%E proteins, ≥30 g fibres per day and 35%E–45%E carbohydrates) on age-related impairment of health within a 36-months RCT conducted in the region of Berlin and Potsdam. 502 eligible men (n = 183) and women (n = 319), aged 50 to 80 years, with an increased risk to develop age-related diseases were randomly assigned to either an intervention group focusing on NutriAct dietary pattern or a control group focusing on usual care and dietary recommendations in accordance to the German Nutrition Society (DGE). In the intervention group, 21 nutrition counsellings as well as supplementation of rapeseed oil, oil cake and specific designed foods are used to achieve the intended NutriAct dietary pattern. The primary outcome is a composite endpoint of age-related disorders, including cardiovascular morbidity, decline of cognitive function as well as clinical features of sarcopenia. Secondary outcomes include diet-induced effects on quality of life, depression, frailty, cardiovascular function, bone density, fat distribution pattern, glucose, lipid and energy metabolism, as well as the identification of biomarkers linked with age-related disorders. Discussion: The findings of this trial will provide clinically relevant information regarding dietary effects on age-related impairment of health and will contribute to the definition of the optimal macronutrient composition in the context of healthy aging in the German population.

The skeletal muscle plays an important role in maintaining whole-body mechanics, metabolic homeostasis, and interorgan crosstalk. However, during aging, functional and structural changes such as fiber integrity loss and atrophy can occur across different species. A commonly observed hallmark of aged skeletal muscle is the accumulation of oxidatively modified proteins and protein aggregates which point to an imbalance in proteostasis systems such as degradation machineries. Recently, we showed that the ubiquitin-proteasomal system was impaired. Specifically, the proteasomal activity, which was declining in aged M. soleus (SOL) and M. extensor digitorum longus (EDL). Therefore, in order to understand whether another proteolytic system would compensate the decline in proteasomal activity, we aimed to investigate age-related changes in the autophagy-lysosomal system (ALS) in SOL, mostly consisting of slow-twitch fibers, and EDL, mainly composed of fast-twitch fibers, from young (4 months) and old (25 months) C57BL/6JRj mice. Here, we focused on changes in the content of modified proteins and the ALS. Our results show that aged SOL and EDL display high levels of protein modifications, particularly in old SOL. While autophagy machinery appears to be functional, lysosomal activity declines gradually in aged SOL. In contrast, in old EDL, the ALS seems to be affected, demonstrated by an increased level of key autophagy-related proteins, which are known to accumulate when their delivery or degradation is impaired. In fact, lysosomal activity was significantly decreased in old EDL. Results presented herein suggest that the ALS can compensate the high levels of modified proteins in the more oxidative muscle, SOL, while EDL seems to be more prone to ALS age-related alterations.

Globally, cardiovascular diseases are the leading cause of death in the aging population. While the clinical pathology of the aging heart is thoroughly characterized, underlying molecular mechanisms are still insufficiently clarified. The aim of the present study was to establish an in vitro model system of cardiomyocyte premature senescence, culturing heart muscle cells derived from neonatal C57Bl/6J mice for 21 days. Premature senescence of neonatal cardiac myocytes was induced by prolonged culture time in an oxygen-rich postnatal environment. Age-related changes in cellular function were determined by senescence-associated β-galactosidase activity, increasing presence of cell cycle regulators, such as p16, p53, and p21, accumulation of protein aggregates, and restricted proteolysis in terms of decreasing (macro-)autophagy. Furthermore, the culture system was functionally characterized for alterations in cell morphology and contractility. An increase in cellular size associated with induced expression of atrial natriuretic peptides demonstrated a stress-induced hypertrophic phenotype in neonatal cardiomyocytes. Using the recently developed analytical software tool Myocyter, we were able to show a spatiotemporal constraint in spontaneous contraction behavior during cultivation. Within the present study, the 21-day culture of neonatal cardiomyocytes was defined as a functional model system of premature cardiac senescence to study age-related changes in cardiomyocyte contractility and autophagy.

Aging is accompanied by the accumulation of cellular damage over time in response to stress, lifestyle and environmental factors ultimately leading to age-related diseases and death. Additionally, the number of senescent cells increases with age. Senescence is most likely not a static endpoint, it represents a series of hallmarks including morphological changes, alterations in protein turnover and accumulation of protein aggregates. The importance of protein oxidation and aggregate accumulation in the progression of aging is not yet fully understood and research to what extent the accumulation of oxidized proteins has an effect on senescence and the aging process is still ongoing. To study the mechanisms of aging, the impact of senescence and the role of protein aggregates on the aging process, cell culture models are useful tools. Most notably stress induced premature senescence (SIPS) models have contributed to the identification of mechanisms involved in the aging process and helped unravel the age-related changes in proteolysis and the importance of protein aggregation. Here we review characteristics of replicative and premature senescence, how to induce most frequently used senescence models and gained knowledge on age-related changes in the major proteolytic systems.

Purpose of review The aim of this article is to present a brief overview of recently published articles assessing oxidative stress markers in nutritional studies. Recent findings Intervention and observational studies were carried out in both, healthy subjects and patients and describe the association of foodstuffs as well as isolated nutrients with biomarkers of oxidative stress. The results from human intervention studies on healthy participants and patients are controversial. Long-term interventions (>8 weeks) seem to be more effective than short-term or single-dose interventions. Results are difficult to compare because not only the methods used, also the assessed biomarkers and outcomes were very diverse. In addition, studies vary in the compounds and doses used, duration, participants and so on. Different biomarkers (damaged molecules together with antioxidants from different compartments) should be assessed to evaluate the true 'redox-status' of an individual and the impact of a nutritional intervention. Summary Both observational and interventional studies performed in healthy participants and patients show possible beneficial effects of nutrients and foodstuffs by improving oxidative stress markers and antioxidant enzyme activities. Biomarkers should be standardized to allow better comparison of results of antioxidant intervention studies.

The production of reactive species is an inevitable by-product of metabolism and thus, life itself. Since reactive species are able to damage cellular structures, especially proteins, as the most abundant macromolecule of mammalian cells, systems are necessary which regulate and preserve a functional cellular protein pool, in a process termed “proteostasis”. Not only the mammalian protein pool is subject of a constant turnover, organelles are also degraded and rebuild. The most important systems for these removal processes are the “ubiquitin-proteasomal system” (UPS), the central proteolytic machinery of mammalian cells, mainly responsible for proteostasis, as well as the “autophagy-lysosomal system”, which mediates the turnover of organelles and large aggregates. Many age-related pathologies and the aging process itself are accompanied by a dysregulation of UPS, autophagy and the cross-talk between both systems. This review will describe the sources and effects of oxidative stress, preservation of cellular protein- and organelle-homeostasis and the effects of aging on proteostasis in mammalian cells., Graphical abstract fx1

Mitochondria have been in the focus of oxidative stress and aging research for decades due to their permanent production of ROS during the oxidative phosphorylation. The hypothesis exists that mitochondria are involved in the formation of lipofuscin, an autofluorescent protein aggregate that accumulates progressively over time in lysosomes of post-mitotic and senescent cells. To investigate the influence and involvement of mitochondria in lipofuscinogenesis, we analyzed lipofuscin amounts as well as the mitochondrial function in young and senescent cells. In addition we used an aging model and Lon protease deficient HeLa cells to investigate the influence of mitochondrial degradation processes on lipofuscin formation.We were able to show that mitophagy is impaired in senescent cells resulting in an increased mitochondrial mass and superoxide formation. In addition, the inhibition of mitochondrial fission leads to increased lipofuscin formation.Moreover, we observed that Lon protease downregulation is linked to a higher lipofuscinogenesis whereas the application of the mitochondrial-targeted antioxidant mitoTEMPO is able to prevent the accumulation of this protein aggregate. Keywords: Lipofuscin, Protein aggregates, Lon protease, Aging, Mitochondria, Oxidative stress

Aging is a complex phenomenon and its impact is becoming more relevant due to the rising life expectancy and because aging itself is the basis for the development of age-related diseases such as cancer, neurodegenerative diseases and type 2 diabetes. Recent years of scientific research have brought up different theories that attempt to explain the aging process. So far, there is no single theory that fully explains all facets of aging. The damage accumulation theory is one of the most accepted theories due to the large body of evidence found over the years. Damage accumulation is thought to be driven, among others, by oxidative stress. This condition results in an excess attack of oxidants on biomolecules, which lead to damage accumulation over time and contribute to the functional involution of cells, tissues and organisms. If oxidative stress persists, cellular senescence is a likely outcome and an important hallmark of aging. Therefore, it becomes crucial to understand how senescent cells function and how they contribute to the aging process. This review will cover cellular senescence features related to the protein pool such as morphological and molecular hallmarks, how oxidative stress promotes protein modifications, how senescent cells cope with them by proteostasis mechanisms, including antioxidant enzymes and proteolytic systems. We will also highlight the nutritional status of senescent cells and aged organisms (including human clinical studies) by exploring trace elements and micronutrients and on their importance to develop strategies that might increase both, life and health span and postpone aging onset., Graphical abstract fx1

Oxidative stress and antioxidants play a role in age-related diseases and in the aging process. We here present data on protein carbonyls, 3-nitrotyrosine, malondialdehyde, and cellular and plasma antioxidants (glutathione, cysteine, ascorbic acid, uric acid,α-tocopherol, and lycopene) and their relation with age in the European multicenter study MARK-AGE. To avoid confounding, only data from countries which recruited subjects from all three study groups (five of eight centers) and only participants aged ≥55 years were selected resulting in data from 1559 participants. These included subjects from (1) the general population, (2) members from long-living families, and (3) their spouses. In addition, 683 middle-aged reference participants (35–54 years) served as a control. After adjustment for age, BMI, smoking status, gender, and country, there were differences in protein carbonyls, malondialdehyde, 3-nitrotyrosine,α-tocopherol, cysteine, and glutathione between the 3 study groups. Protein carbonyls and 3-nitrotyrosine as well as cysteine, uric acid, and lycopene were identified as independent biomarkers with the highest correlation with age. Interestingly, from all antioxidants measured, only lycopene was lower in all aged groups and from the oxidative stress biomarkers, only 3-nitrotyrosine was increased in the descendants from long-living families compared to the middle-aged control group. We conclude that both lifestyle and genetics may be important contributors to redox biomarkers in an aging population.

Changes in the two main intracellular degradation systems, the Ubiquitin-Proteasome System and the Autophagy-Lysosome pathway (ALP) are widely discussed as a hallmark of the aging process. To follow the age-related behavior of both degradation systems we examined their impact on ferritin, known to be degradable by both. Ferritin H was analyzed in young and senescent human fibroblasts, revealing a higher steady-state level in the senescent cells. By blocking both proteolytic systems, we confirmed that particularly the ALP plays a crucial role in ferritin H turnover. However, an unexpected increase in lysosomal activity in the senescent cells, suggests a dysregulation in the autophagy pathway. To further investigate the impaired ferritin H turnover, confocal microscopic colocalization studies of ferritin H with lysosomal-associated membrane protein 2a (Lamp2a) and monodansylcadaverine (MDC) were performed and clearly revealed the degradation of ferritin by macroautophagy. By induction of autophagy via inhibition of mTOR using rapamycin an increase of ferritin H turnover was obtained in senescent cells, demonstrating a mTOR dependent reduction of autophagy in senescent human fibroblasts.

Context Meal timing affects metabolic homeostasis and body weight, but how composition and timing of meals affect plasma lipidomics in humans is not well studied. Objective We used high throughput shotgun plasma lipidomics to investigate effects of timing of carbohydrate and fat intake on lipid metabolism and its relation to glycemic control. Design 29 nondiabetic men consumed (1) a high-carb test meal (MTT-HC) at 09.00 and a high-fat meal (MTT-HF) at 15.40; or (2) MTT-HF at 09.00 and MTT-HC at 15.40. Blood was sampled before and 180 minutes after completion of each MTT. Subcutaneous adipose tissue (SAT) was collected after overnight fast and both MTTs. Prior to each investigation day, participants consumed a 4-week isocaloric diet of the same composition: (1) high-carb meals until 13.30 and high-fat meals between 16.30 and 22:00 or (2) the inverse order. Results 12 hour daily lipid patterns showed a complex regulation by both the time of day (67.8%) and meal composition (55.4%). A third of lipids showed a diurnal variation in postprandial responses to the same meal with mostly higher responses in the morning than in the afternoon. Triacylglycerols containing shorter and more saturated fatty acids were enriched in the morning. SAT transcripts involved in fatty acid synthesis and desaturation showed no diurnal variation. Diurnal changes of 7 lipid classes were negatively associated with insulin sensitivity, but not with glucose and insulin response or insulin secretion. Conclusions This study identified postprandial plasma lipid profiles as being strongly affected by meal timing and associated with insulin sensitivity.

Recently, Weber et al. published a thorough investigation of the age-dependency of oxidative stress (OS) determined by the steady state concentrations of different compounds - oxidation products and antioxidants - that are in common use as biomarkers of OS in 2207 healthy individuals of the cross-sectional MARK-AGE Project. The correlations among biomarkers were significant but weak. These findings may indicate different manifestations of OS and must further be evaluated. Here, we report a refined analysis of OS based on the above-mentioned original data. We show that malondialdehyde (MDA) appears to be sensitive to both gender and age. It is significantly lower and shows a greater age-dependence in women than in men. The age-dependency of MDA in women arises in a stepwise fashion. The age-dependent slope of the steady state concentration is maximal at the age between 50 and 55 years, indicating that it may be attributed to the change of metabolism in the post-menopause. Interestingly, total glutathione (GSH) decreased with age simultaneously with the increase in MDA.Different biomarkers yield different gender- and age-dependencies. Unlike the concentration of MDA, the concentrations of the other two oxidation products, i.e. protein carbonyls and 3-nitrotyrosine were similar in men and women and appeared to be independent of age in the healthy study population. The analyzed antioxidants exhibited different gender- and age-dependencies. In conclusion, it appears that all the biomarkers assessed here reflect different types of OS and that MDA and GSH reflect the same type of OS. Keywords: Oxidative stress, Age-dependency, Gender-associated differences, Biomarkers

Aging is accompanied by the accumulation of oxidized proteins. To remove them, cells employ the proteasomal and autophagy-lysosomal systems; however, if the clearance rate is inferior to its formation, protein aggregates form as a hallmark of proteostasis loss. In cells, during stress conditions, actin aggregates accumulate leading to impaired proliferation and reduced proteasomal activity, as observed in cellular senescence. The heat shock protein 90 (Hsp90) is a molecular chaperone that binds and protects the proteasome from oxidative inactivation. We hypothesized that in oxidative stress conditions a malfunction of Hsp90 occurs resulting in the aforementioned protein aggregates. Here, we demonstrate that upon oxidative stress Hsp90 loses its function in a highly specific non-enzymatic iron-catalyzed oxidation event and its breakdown product, a cleaved form of Hsp90 (Hsp90cl), acquires a new function in mediating the accumulation of actin aggregates. Moreover, the prevention of Hsp90 cleavage reduces oxidized actin accumulation, whereas transfection of the cleaved form of Hsp90 leads to an enhanced accumulation of oxidized actin. This indicates a clear role of the Hsp90cl in the aggregation of oxidized proteins., Graphical abstract fx1

Dietary methionine restriction (MR) is well known to reduce body weight by increasing energy expenditure (EE) and insulin sensitivity. An elevated concentration of circulating fibroblast growth factor 21 (FGF21) has been implicated as a potential underlying mechanism. The aims of our study were to test whether dietary MR in the context of a high-fat regimen protects against type 2 diabetes in mice and to investigate whether vegan and vegetarian diets, which have naturally low methionine levels, modulate circulating FGF21 in humans. New Zealand obese (NZO) mice, a model for polygenic obesity and type 2 diabetes, were placed on isocaloric high-fat diets (protein, 16 kcal%; carbohydrate, 52 kcal%; fat, 32 kcal%) that provided methionine at control (Con; 0.86% methionine) or low levels (0.17%) for 9 wk. Markers of glucose homeostasis and insulin sensitivity were analyzed. Among humans, low methionine intake and circulating FGF21 levels were investigated by comparing a vegan and a vegetarian diet to an omnivore diet and evaluating the effect of a short-term vegetarian diet on FGF21 induction. In comparison with the Con group, MR led to elevated plasma FGF21 levels and prevented the onset of hyperglycemia in NZO mice. MR-fed mice exhibited increased insulin sensitivity, higher plasma adiponectin levels, increased EE, and up-regulated expression of thermogenic genes in subcutaneous white adipose tissue. Food intake and fat mass did not change. Plasma FGF21 levels were markedly higher in vegan humans compared with omnivores, and circulating FGF21 levels increased significantly in omnivores after 4 d on a vegetarian diet. These data suggest that MR induces FGF21 and protects NZO mice from high-fat diet–induced glucose intolerance and type 2 diabetes. The normoglycemic phenotype in vegans and vegetarians may be caused by induced FGF21. MR akin to vegan and vegetarian diets in humans may offer metabolic benefits via increased circulating levels of FGF21 and merits further investigation.—Castaño-Martinez, T., Schumacher, F., Schumacher, S., Kochlik, B., Weber, D., Grune, T., Biemann, R., McCann, A., Abraham, K., Weikert, C., Kleuser, B., Schürmann, A., Laeger, T. Methionine restriction prevents onset of type 2 diabetes in NZO mice.

To examine the reaction of microglial cells (MG) when incubated with lipofuscin (LP) in vitro with emphasis on the immunological reaction of the MG toward LP and the suppression of this reaction by immunomodulatory agents. MG are involved in the pathogenesis of degenerative eye disorders such as age-related macular degeneration (AMD). LP is a heterogeneous waste material that accumulates in the retinal pigment epithelium (RPE) cells with advancing age. LP is known to have toxic effects on RPE cells and therefore an elevated LP-derived fundus autofluorescence is a risk factor for AMD development. MG in the subretinal space have been reported in eyes affected by AMD. Moreover, in senescent mice, subretinal MG were found, which display an autofluorescence that may be derived from LP uptake. In this study, we incubated MG (BV-2 cell line and primary cells from murine brain) in vitro with LP isolated from the human RPE. We observed phagocytosis, studied cell morphologies, and analyzed the cell culture supernatants. We also investigated the effect of the immunomodulatory agents hydrocortisone (HC), minocycline, and the tripeptide TKP. The MG phagocytosed the LP quickly and completely. We detected highly elevated levels of pro-inflammatory cytokines (especially of IL-6, IL-23p19, TNF-α, KC, RANTES, and IL-1α) in the cell culture supernatants. Furthermore, levels of vascular endothelial growth factor (VEGF) were raised in BV-2 cells. Anti-inflammatory agents added to the cell cultures inhibited the inflammatory reaction, in particular hydrocortisone (HC). Minocycline and TKP had less impact on the cytokine release. The interaction of MG and LP could play a role in the development of retinal degeneration by triggering an inflammatory reaction and angiogenesis.

Metallothionein (MT) family are cysteine-rich proteins that regulate zinc (Zn) homeostasis and protect against oxidative damage. Studies in transgenic mice have shown that MT favorably influence longevity, although their role in human aging is not completely understood. Within the European multicenter study MARK-AGE, we analyzed MT induction after Zn treatment in peripheral blood mononuclear cells (PBMCs) and its relation with redox biomarkers in 2,936 age-stratified subjects (35–75 years) including the general population (RASIG), centenarian offspring (GO), and their spouses (SGO). We found that the lymphocyte capability to induce MT in response to Zn is not affected by aging. However, GO participants showed lower Zn-induced MT and increased basal expression of MT1A, MT1X, and ZnT-1 genes than RASIG subjects. Moreover, Zn-induced MT levels were found to be inversely related with oxidative stress markers (plasma protein carbonyls, 3-nitrotyrosine, and malondialdehyde) in the whole population, but not in GO subjects. In conclusion, our results support the hypothesis that the response to Zn is attenuated in PBMCs of centenarian offspring compared to the general population as a consequence of a tighter control of Zn homeostasis which is likely to provide them constant protection against stress stimuli over the whole lifespan. published

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection. Electronic supplementary material The online version of this article (10.1007/s00204-018-2279-8) contains supplementary material, which is available to authorized users.

Due to their bioactivity and harmful potential, advanced glycation end products (AGEs) are discussed to affect human health. AGEs are compounds formed endogenously in the human body andexogenously, especially, in foods while thermal processing. In contrast to endogenous AGEs, dietary AGEs are formed in much higher extent. However, their risk potential is also depending on absorption, distribution, metabolism and elimination. For over 10 years an intense debate on the risk of dietary AGEs on human health is going on. On the one hand, studies provided evidence that dietary AGEs contribute to clinical outcomes. On the other hand, human studies failed to observe any association. Because it was not possible to draw a final conclusion, the call for new interdisciplinary approaches arose. In this review, we will give an overview on the current state of scientific knowledge in this field. In particular, we focus on (I) the occurrence of AGEs in foods and the daily uptake of AGEs, (II) contribution to endogenous levels and (III) the effect on health-/disease-related biomarkers in humans.

International audience; Purpose: We investigated the cross-sectional associations between macular pigment optical density (MPOD), plasma lutein (L), and zeaxanthin (Z) concentrations and cognitive function in 184 older adults of the 3-City-Bordeaux cohort. Methods: MPOD was measured using the two-wavelength autofluorescence method with a modified scanning laser ophthalmoscope. Plasma L and Z (L+Z) concentrations were determined by high-performance liquid chromatography and were considered either crude or expressed as a ratio of the concentration of plasma lipids (total cholesterol [TC] + triglycerides [TG]). Cognitive performances were assessed using the following four separate neuropsychological tests: the Mini-Mental State Examination (MMSE), the Isaacs Set Test (IST), the Benton Visual Retention Test (BVRT), and the sum of the three free recalls of the Free and Cued Selective Reminding Test (FCSRT). These test results were summarized by a composite global cognitive z-score. Results: Higher MPOD at 0.5 degrees was significantly associated with a higher composite z-score (beta = 0.15, 95% confidence interval [CI] 0.04-0.26), higher BVRT (beta = 0.39, 95%CI 0.08-0.70), and higher IST (beta = 1.16, 95%CI 0.11-2.22) performances. Higher plasma L+Z concentrations were significantly associated with higher IST scores (beta = 0.97, 95%CI 0.01-1.94). Furthermore, a higher L+Z/TC+TG ratio was associated with a higher composite z-score (beta = 0.12, 95%CI 0.01-0.23), along with higher IST (beta = 1.02, 95%CI 0.002-2.04) and FCSRT (beta = 1.55, 95%CI 0.41-2.69) performances. Conclusions: This analysis suggested that both higher MPOD and L+Z concentrations were significantly associated with higher cognitive performances. However, MPOD measurements have the advantage of being a fast and representative measure of long-term carotenoid intake.

Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2 alpha pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging.

Background A diet in which fat is mainly eaten in the morning and carbohydrates mainly in the evening (compared with the reverse order) was recently shown to worsen glycemic control in people with prediabetes. Objective We investigated the effects of these dietary patterns on energy metabolism, and on the daily profiles of circulating lipids, adipokines, and inflammatory markers. Design In a randomized controlled crossover trial, 29 nonobese men (with normal glucose tolerance, n = 18; or impaired fasting glucose/glucose tolerance, n = 11) underwent 2 isocaloric 4-wk diets: 1) carbohydrate-rich meals until 1330 and fat-rich meals between 1630 and 2200 (HC/HF); or 2) the inverse sequence of meals (HF/HC). During a 12-h clinical investigation day after each intervention period, 2 meal tolerance tests were performed, at 0900 and 1540, respectively. Substrate oxidation and concentrations of circulating lipids, adipokines, and cytokines were assessed pre- and postprandially. The postprandial inflammatory response in leukocytes was analyzed ex vivo. Results Fasting carbohydrate oxidation decreased (P = 0.004) and lipid oxidation increased (P = 0.012) after the HC/HF diet. Fasting concentrations of blood markers did not differ between diets. The diets modulated the daily profiles of carbohydrate oxidation, lipid oxidation, and β-hydroxybutyrate, although the average daily values of these parameters showed no difference between the diets, and no interaction between diet and glucose tolerance status. Diurnal patterns of triglycerides, low-density lipoprotein cholesterol, leptin, visfatin, and of LPS-induced cytokine secretion in blood leukocytes were also modulated by the diets. Average daily concentrations of leptin (P = 0.017) and visfatin (P = 0.041) were lower on the HF/HC diet than on the HC/HF diet. Conclusions Diurnal distribution of carbohydrates and fat affects the daily profiles of substrate oxidation, circulating lipids, and cytokine secretion, and alters the average daily concentrations of adipokine secretion in nonobese nondiabetic humans. The study was registered at clinicaltrials.gov as NCT02487576.

Association of oxidative stress with carcinogenesis is well known, but not understood well, as is pathophysiology of oxidative stress generated during different types of anti-cancer treatments. Moreover, recent findings indicate that cancer associated lipid peroxidation might eventually help defending adjacent nonmalignant cells from cancer invasion. Therefore, untargeted metabolomics studies designed for advanced translational and clinical studies are needed to understand the existing paradoxes in oncology, including those related to controversial usage of antioxidants aiming to prevent or treat cancer. In this short review we have tried to put emphasis on the importance of pathophysiology of oxidative stress and lipid peroxidation in cancer development in relation to metabolic adaptation of particular types of cancer allowing us to conclude that adaptation to oxidative stress is one of the main driving forces of cancer pathophysiology. With the help of metabolomics many novel findings are being achieved thus encouraging further scientific breakthroughs. Combined with targeted qualitative and quantitative methods, especially immunochemistry, further research might reveal bio-signatures of individual patients and respective malignant diseases, leading to individualized treatment approach, according to the concepts of modern integrative medicine. Keywords: Carcinogenesis, Cancer, Oxidative stress, Lipid peroxidation, 4-hydroxynonenal, Glutathione, Metabolomics, Immunochemistry, Biomarkers, Omics science

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions., DFG (German Research Foundation), by Spanish Government grants PI15/00107 and PT13/0001/0024 (partially funded by the European Union ERDF)