Moderation: J. Haendeler, Düsseldorf; J. Altschmied, Düsseldorf
Background and objective: The hormone cortisol plays a crucial role in many metabolic pathways. Cortisol secretion follows a circadian rhythm characterized by high concentrations in the morning and low concentrations at midnight. Moreover, a circannual rhythm in cortisol secretion has been reported for younger adults while no data exist from older cohorts. Since aging is characterized by a general decline in rhythmicity, this study aimed to analyze whether seasonal variation in cortisol concentrations is still present in older people.
Subjects and methods: Participants were 67 healthy non-smoking older women (69.2 ± 5.5 yrs) and 56 young adult women (24.3 ± 3.5 yrs) for comparison. Salivary samples were taken in summer (35 older, 37 young women) and in winter (32 older, 19 young women). Cortisol concentrations were quantified in duplicate from the salivary samples, collected at 0 min (T0), 30 min (T30), and 60 min (T60) post awakening and in the evening before bedtime. Derived values were morning increase in cortisol concentration (cortisol awakening response, T30 minus T0) and diurnal decline in cortisol concentration (T30 minus evening cortisol). For seasonal comparisons, t-tests were applied.
Results: Both older and young women displayed higher cortisol concentrations in winter compared with summer at T0, T30, T60 and in the evening. Differences in cortisol concentrations between winter and summer were significant in older women at T30 (33.4 ± 11.4 vs. 25.9 ± 11.5 nmol/L, p = 0.013) and at T60 (26.6 ± 12.6 vs. 19.6 ± 8.3 nmol/L, p = 0.020). Neither older nor young women showed seasonal differences in their diurnal pattern, i.e. in the morning increase and diurnal decline of cortisol concentrations (p > 0.050). Further, there were no age group differences in cortisol values between older and young women, neither in winter nor in summer (p > 0.050).
Conclusions: The observed seasonal variation in cortisol concentrations suggests that the circannual rhythm in cortisol secretion, found in younger adults, is still present in older ones. The absence of seasonal differences in the diurnal pattern of cortisol concentration (morning increase and diurnal decline) indicates maintenance of diurnal profile from summer to winter in older women.
Clinical studies showed that the mineralocorticoid receptor (MR), a ligand-dependent transcription factor, mediates pathophysiological processes like inflammation and endothelial dysfunctions in the cardiovascular system. Interestingly, the MR and its natural ligand aldosterone per se are not capable of eliciting pathophysiological effects but require a certain micro-milieu. Several studies indicate that a parainflammatory micro-milieu created by altered level of oxidative and nitrosative stress represents a possible trigger to induce pathophysiological MR effects. During ageing, vascular cells are increasingly exposed to such a parainflammatory micro-milieu, which could then augment pathophysiological MR signaling and lead to age-associated changes. Our previous investigations showed that genomic MR activity is influenced by nitrosative stress. We found that the nitric oxide (NO) donor SNAP can attenuate aldosterone-induced transcriptional MR activity by reducing its binding to DNA elements. Additionally, the peroxynitrite (ONOO–) donor SIN-1 acted as a ligand-independent MR activator. To investigate whether amino acids of the MR are directly posttranslationally modified by nitrosative stress, we performed co-immunoprecipitation and Western blot experiments. Thereby we could show an increased MR S-nitrosylation level after NO treatment whereas ONOO– stimulation led to an elevated amount of nitrated MR tyrosines. Mass spectrometry analyses after biotin switch identified C639 and C663 as S-nitrosylated MR residues. C663 was generally S-nitrosylated whereas C639 was only S-nitrosylated after NO treatment. To analyze the importance of these S-nitrosylated MR residues for MR function, site-directed mutagenesis was performed by conversion of cysteine into serine (S). Subsequently, the transactivation activity of mutated MR was compared to wild type MR (WT MR) in reporter gene assays. MR C663S possessed an unchanged basal and maximum transactivation activity, but had a lower EC50 value than WT MR. MR C639S exhibited a reduced basal and maximal transactivation but had the same aldosterone inducibility as the WT MR. The effect of SNAP on the transcriptional activity of MR C639S compared to WT MR was reduced by trend. Overall, our data show that cysteine 639 and 663 of the MR are directly modified under nitrosative stress conditions, thereby altering basal as well as aldosterone-induced genomic MR activity and NO responsiveness.
Advanced glycation end products (AGEs) play important role in senescence and age-related diseases by impairing protein structure and functions. These molecules can interact with receptors to AGEs (RAGE) and activate numerous pro-inflammatory and pro-fibrogenic signaling pathways.
The aim of the work is to evaluate removal of AGE-modified proteins from human plasma, using polymer adsorbent system, and to identify cellular responses towards the retained proteins.
For this aim, a column material from a medical device, used for continuous hemofiltration in sepsis patient, was eluted with denaturing buffer. Amount of eluted protein was quantified by slot-blot, along with albumin standards to obtain standard curve. Posttranslational protein modifications were identified via immunoblotting.
Carboxymethyllysine, argpyrimidine, methylglyoxal-derived hydroimidazolone-1, pentosidine, and carbonyls were identified in eluted plasma proteins. Human umbilical vein endothelial cells (HUVEC) were stimulated with 10, 50, 100, 500 and 1000 ng of eluted proteins. After 24 hours of stimulation, cells were harvested. Stimulation of HUVECs with 10 ng elution resulted in increased expression of RAGE, compared to non-stimulated cells. Trends towards NF-?B and p38MAPK activation in response to 1-day treatment were observed, indicating a proinflammatory potential of isolated proteins.
Thus, adsorbed modified proteins induce increase in RAGE expression in HUVECs and activate downstream targets of the RAGE-signaling pathways. Further identification of cytotoxic effects and influence of the isolated modified proteins on cell cycle is required to better understand the physiological effect of repeated hemofiltration procedures in patients.
Advanced Glycation Endproducts (AGE) are formed when reducing sugars react with proteins. These modifications alter the spatial orientation of amino acid residues as a consequence of which changes in the structure and function of proteins occur. The modified proteins can accumulate in different tissues and cause a reduction in organ function. One of the best known examples for this is the accumulation of AGEs in the Extracellular Matrix causing increased stiffness and decreased elasticity in arteries and the ventricles of the heart leading to diastolic heart insufficiency and hypertension.
Lately it has also become known that the immune system reacts to the presence of AGE modified proteins by producing autoantibodies against them. Autoantibodies against glycated histones are discussed to play a major role in autoimmune disease such as systemic lupus erythematosus, rheumatoid arthritis and diabetes.
The goal of the project is to better understand the role of these autoantibodies in health and disease. An ELISA was developed in order to detect the autoantibodies in serum samples. Serum samples from patients undergoing Cardiac Artery Bypass Grafting (CABG) in the University Clinic of Halle were analysed to understand, if levels of these autoantibodies may correlate with the morbidity outcome.
Obesity and inflammation are recognized factors contributing to the pathogenesis of sarcopenia, which is characterized by a loss of skeletal muscle mass, strength, and function. Intramuscular fat deposits were associated with compromised muscle integrity; however, the relevant fat compounds and their roles as mediators of muscular inflammation are not known. The aim of this study was to identify potential correlations between inflammation markers and lipid compounds that accumulate in the quadriceps muscle of aged Sprague-Dawley (SD) rats, a gender-specific model of sarcopenic obesity. Six-month-old SD rats were continuously fed a control (CD) or high-fat diet (HFD) until the age of 21 months. Histological assessment, magnetic resonance imaging (MRI) and gas chromatography-mass spectrometry (GC-MS) were used for a comprehensive analysis of tissue integrity and lipid content in rats’ quadriceps muscles. Our results show the onset of sarcopenia in quadriceps muscles of 21-months-old male SD rats exclusively, which was further enhanced by the long-term consumption of a high-fat diet (HFD). This trend is accompanied by accumulation of long-chain fatty acids, like octadecadienoic, octadecanoic and octadecenoic acid esters in quadriceps muscles of male rats, but not in female rats. The designated fatty acids are mainly incorporated into triacylglycerols (TAG) or free fatty acids (FFAs), and their proportions are significantly elevated by consumption of a HFD. Further multiplex RNA and protein arrays of 22 prominent inflammation markers indicate a significant increase of the RANTES, MCP-1 and MIP-2 chemokines in quadriceps muscle tissue of HFD-fed male rats, but not female rats. Hence, the quadriceps muscles of 21-months-old male SD rats comprise a slightly higher number of resident immune cells compared to female rats. In conclusion, our study suggests that sarcopenia is associated with greater deposits of long-chain fatty acid esters and increased levels of the inflammatory markers RANTES, MCP-1 and MIP-2 in skeletal muscles of old male SD rats. This trend is further reinforced by long-term consumption of a HFD, which may provoke a synergistic crosstalk between long-chain fatty acids and inflammatory pathways in sarcopenic muscle.
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