Chain-length dependent effects of inorganic polyphosphate on endothelial function and nucleotide pool.

Endothelial cells (ECs) are the first line that comes into contact with blood pathogens, pathogen-derived molecules, and factors that stimulate coagulation and inflammation. Inorganic polyphosphate (polyP) - a polymer of orthophosphate units synthesized by bacteria under stress and released by platelets upon their activation is among these factors. Bacterial and platelet polyPs differ in length, and both variants elicit different effects in eukaryotes. This study aimed to investigate how bacterial-like long-chain polyP (Lc-polyP) and platelet-like short-chain polyP (Sc-polyP) affect the functionality of cultured endothelial cells. Murine immortalized heart endothelial cells (H5V) were exposed to polyP of different chain lengths to assess the effects of these stimuli on intracellular energetics, permeability, and endothelial adhesion. We observed varying effects between Lc-polyP and Sc-polyP treatments. Lc-polyP more potently disturbs the intracellular ATP pool, a parameter strongly connected with vascular injury, whereas Sc-polyP robustly stimulates cellular adhesion to the endothelium. Both polymers similarly enhance endothelial permeability, suggesting potent immunomodulatory properties. This study provides evidence that polyP elicits profound cellular responses in endothelium depending on the polymer's length.

Seasonality and Objective Physical Activity and Sedentary Behaviour among Older Adults from Four European Countries.

OBJECTIVE: The present study aimed to explore the relationship between objective physical activity and sedentary behaviour with seasonality among a sample of older adults living in four European countries. METHODS: A sample of 169 older adults living in Croatia, Greece, Portugal, and Poland (mean age = 72.2 ± 6.0, 68% female) had valid objective physical activity and sedentary behaviour data collected in different seasons of the year: spring and autumn/winter. Physical activity and sedentary behaviour were collected with accelerometers (ActiGraph, GT3X), over 7 consecutive days, in both periods. A valid record was defined as at least two weekdays and one weekend day with 10 hours of wearing time. Analyses were performed with IBM SPSS 28.0, using t-test, ANOVA, and binary logistic regressions. RESULTS: Most older adults from the four countries met the physical activity guidelines in spring and autumn/winter. No significant variations were found across seasons for sedentary behaviour and physical activity both for light and vigorous intensity, regardless of sex, country, education, and body mass index (BMI). A decline in moderate physical activity intensity from spring to autumn/winter was found for those with lower education and higher BMI. CONCLUSION: The promotion of physical activity must be considered in programs to promote healthy aging throughout the year, especially considering the moderate intensity and those populations with higher BMI and lower educational levels.

Metabolomic Footprint of Disrupted Energetics and Amino Acid Metabolism in Neurodegenerative Diseases: Perspectives for Early Diagnosis and Monitoring of Therapy.

The prevalence of neurodegenerative diseases (NDs) is increasing due to the aging population and improved longevity. They are characterized by a range of pathological hallmarks, including protein aggregation, mitochondrial dysfunction, and oxidative stress. The aim of this review is to summarize the alterations in brain energy and amino acid metabolism in Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Based on our findings, we proposed a group of selected metabolites related to disturbed energy or mitochondrial metabolism as potential indicators or predictors of disease. We also discussed the hidden challenges of metabolomics studies in NDs and proposed future directions in this field. We concluded that biochemical parameters of brain energy metabolism disruption (obtained with metabolomics) may have potential application as a diagnostic tool for the diagnosis, prediction, and monitoring of the effectiveness of therapies for NDs. However, more studies are needed to determine the sensitivity of the proposed candidates. We suggested that the most valuable biomarkers for NDs studies could be groups of metabolites combined with other neuroimaging or molecular techniques. To attain clinically applicable results, the integration of metabolomics with other "omic" techniques might be required.

Rosiglitazone Ameliorates Cardiac and Skeletal Muscle Dysfunction by Correction of Energetics in Huntington's Disease.

Huntington's disease (HD) is a rare neurodegenerative disease that is accompanied by skeletal muscle atrophy and cardiomyopathy. Tissues affected by HD (central nervous system [CNS], skeletal muscle, and heart) are known to suffer from deteriorated cellular energy metabolism that manifests already at presymptomatic stages. This work aimed to test the effects of peroxisome proliferator-activated receptor (PPAR)-γ agonist-rosiglitazone on grip strength and heart function in an experimental HD model-on R6/1 mice and to address the mechanisms. We noted that rosiglitazone treatment lead to improvement of R6/1 mice grip strength and cardiac mechanical function. It was accompanied by an enhancement of the total adenine nucleotides pool, increased glucose oxidation, changes in mitochondrial number (indicated as increased citric synthase activity), and reduction in mitochondrial complex I activity. These metabolic changes were supported by increased total antioxidant status in HD mice injected with rosiglitazone. Correction of energy deficits with rosiglitazone was further indicated by decreased accumulation of nucleotide catabolites in HD mice serum. Thus, rosiglitazone treatment may not only delay neurodegeneration but also may ameliorate cardio- and myopathy linked to HD by improvement of cellular energetics.

4-Pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR)-A Novel Oncometabolite Modulating Cancer-Endothelial Interactions in Breast Cancer Metastasis.

The accumulation of specific metabolic intermediates is known to promote cancer progression. We analyzed the role of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR), a nucleotide metabolite that accumulates in the blood of cancer patients, using the 4T1 murine in vivo breast cancer model, and cultured cancer (4T1) and endothelial cells (ECs) for in vitro studies. In vivo studies demonstrated that 4PYR facilitated lung metastasis without affecting primary tumor growth. In vitro studies demonstrated that 4PYR affected extracellular adenine nucleotide metabolism and the intracellular energy status in ECs, shifting catabolite patterns toward the accumulation of extracellular inosine, and leading to the increased permeability of lung ECs. These changes prevailed over the direct effect of 4PYR on 4T1 cells that reduced their invasive potential through 4PYR-induced modulation of the CD73-adenosine axis. We conclude that 4PYR is an oncometabolite that affects later stages of the metastatic cascade by acting specifically through the regulation of EC permeability and metabolic controls of inflammation.

Differences in Extracellular NAD+ and NMN Metabolism on the Surface of Vascular Endothelial Cells.

The disruption of the metabolism of extracellular NAD+ and NMN may affect related signaling cascades and pathologies, such as cardiovascular or respiratory system diseases. We aimed to study NAD+ and NMN hydrolysis on surface endothelial cells of diverse origins and with genetically modified nucleotide catabolism pathways. We tested lung endothelial cells isolated from C57BL/6 J wild-type (WT) and C57BL/6 J CD73 knockout (CD73 KO) mice, the transfected porcine iliac artery endothelial cell line (PIEC) with the human E5NT gene for CD73 (PIEC CD73), and a mock-transfected control (PIEC MOCK), as well as HMEC-1 and H5V cells. Substrate conversion into the product was followed by high-performance liquid chromatography (HPLC). We showed profound differences in extracellular NAD+ and NMN metabolism related to the vessel origin, species diversity, and type of culture. We also confirmed the involvement of CD38 and CD73 in NAD+ and NMN cleavage.

Inorganic Polyphosphate-Regulator of Cellular Metabolism in Homeostasis and Disease.

Inorganic polyphosphate (polyP), a simple anionic polymer consisting of even hundreds of orthophosphate units, is a universal molecule present in both simple and complex organisms. PolyP controls homeostatic processes in animals, such as blood coagulation, tissue regeneration, and energy metabolism. Furthermore, this polymer is a potent regulator of inflammation and influences host immune response in bacterial and viral infections. Disturbed polyP systems have been related to several pathological conditions, including neurodegeneration, cardiovascular disorders, and cancer, but we lack a full understanding of polyP biogenesis and mechanistic insights into the pathways through which polyP may act. This review summarizes recent studies that describe the role of polyP in cell homeostasis and show how disturbances in polyP levels may lead to disease. Based on the collected findings, we highlight the possible usage of this polymer as a promising therapeutic tool in multiple pathologies.

Huntingtin protein maintains balanced energetics in mouse cardiomyocytes.

Huntingtin (HTT) is a multifunctional protein crucial for proper embryogenesis and nervous system development. Mutation of a single allele in gene coded this protein results in the Huntington׳s disease (HD). There is growing evidence of cardiovascular system pathologies coexisting with the neurological symptoms in HD patients. Thus, this study aims to establish the role of huntingtin protein in cardiomyocytes cellular energy and nucleotides metabolism. We used HTT KO mice embryonic stem cells (ESC) obtained with CRISPR method, wild type mice ESC treated by CRISPR with Scramble control sequence (SCR) as well as wild type (WT) mice ESC and differentiate it into cardiomyocytes. Analysis of intracellular concentration of ATP, ADP, and NAD+, as well as nucleotide catabolites were performed with HPLC. We noted that HTT null cardiomyocytes showed diminished intracellular ATP (4.9 ± 0.5; 6.7 ± 0.4 nmol/mg protein HTT KO vs. SCR) and NAD+ (0.9 ± 0.1; 1.6 ± 0.1 nmol/mg HTT KO vs. SCR). We noted also reduced cellular medium concentration of total purines pool (17.1 ± 1.7; 24.7 ± 2.7 nmol/ml HTT KO vs. SCR) as well as IMP concentration (7.7 ± 0.6; 10.2 ± 0.4 nmol/ml HTT KO vs. SCR). This study indicates that HTT plays an important role in cellular energy balance as well as in nucleotide metabolism in cardiomyocytes. Furthermore, our findings underline that the deterioration in energy metabolism observed in HD may be caused not only by cellular mutant HTT accumulation but also by the loss of HTT function.

Enhanced Muscle Strength in Dyslipidemic Mice and Its Relation to Increased Capacity for Fatty Acid Oxidation.

Dyslipidemia is commonly linked to skeletal muscle dysfunction, accumulation of intramyocellular lipids, and insulin resistance. However, our previous research indicated that dyslipidemia in apolipoprotein E and low-density lipoprotein receptor double knock-out mice (ApoE/LDLR -/-) leads to improvement of exercise capacity. This study aimed to investigate in detail skeletal muscle function and metabolism in these dyslipidemic mice. We found that ApoE/LDLR -/- mice showed an increased grip strength as well as increased troponins, and Mhc2 levels in skeletal muscle. It was accompanied by the increased skeletal muscle mitochondria numbers (judged by increased citrate synthase activity) and elevated total adenine nucleotides pool. We noted increased triglycerides contents in skeletal muscles and increased serum free fatty acids (FFA) levels in ApoE/LDLR -/- mice. Importantly, Ranolazine mediated inhibition of FFA oxidation in ApoE/LDLR -/- mice led to the reduction of exercise capacity and total adenine nucleotides pool. Thus, this study demonstrated that increased capacity for fatty acid oxidation, an adaptive response to dyslipidemia leads to improved cellular energetics that translates to increased skeletal muscle strength and contributes to increased exercise capacity in ApoE/LDLR -/- mice.

High Throughput Procedure for Comparative Analysis of In Vivo Cardiac Glucose or Amino Acids Use in Cardiovascular Pathologies and Pharmacological Treatments.

The heart is characterized by the prominent flexibility of its energy metabolism and is able to use diverse carbon substrates, including carbohydrates and amino acids. Cardiac substrate preference could have a major impact on the progress of cardiac pathologies. However, the majority of methods to investigate changes in substrates' use in cardiac metabolism in vivo are complex and not suitable for high throughput testing necessary to understand and reverse these pathologies. Thus, this study aimed to develop a simple method that would allow for the analysis of cardiac metabolic substrate use. The developed methods involved the subcutaneous injection of stable 13C isotopomers of glucose, valine, or leucine with mass spectrometric analysis for the investigation of its entry into cardiac metabolic pathways that were deducted from 13C alanine and glutamate enrichments in heart extracts. The procedures were validated by confirming the known effects of treatments that modify glucose, free fatty acids, and amino acid metabolism. Furthermore, we studied changes in the energy metabolism of CD73 knock-out mice to demonstrate the potential of our methods in experimental research. The methods created allowed for fast estimation of cardiac glucose and amino acid use in mice and had the potential for high-throughput analysis of changes in pathology and after pharmacological treatments.

Purine Nucleotides Metabolism and Signaling in Huntington's Disease: Search for a Target for Novel Therapies.

Huntington's disease (HD) is a multi-system disorder that is caused by expanded CAG repeats within the exon-1 of the huntingtin (HTT) gene that translate to the polyglutamine stretch in the HTT protein. HTT interacts with the proteins involved in gene transcription, endocytosis, and metabolism. HTT may also directly or indirectly affect purine metabolism and signaling. We aimed to review existing data and discuss the modulation of the purinergic system as a new therapeutic target in HD. Impaired intracellular nucleotide metabolism in the HD affected system (CNS, skeletal muscle and heart) may lead to extracellular accumulation of purine metabolites, its unusual catabolism, and modulation of purinergic signaling. The mechanisms of observed changes might be different in affected systems. Based on collected findings, compounds leading to purine and ATP pool reconstruction as well as purinergic receptor activity modulators, i.e., P2X7 receptor antagonists, may be applied for HD treatment.

Hair dysmorphology in the R6/1 and R6/2 mouse models of Huntington's disease.

Huntington's disease (HD), caused by expansion of CAG repeats in the 1st exon of the HTT gene, is a disorder inherited in an autosomal dominant manner. HD symptoms include chorea, behavioral disturbances and cognitive decline. Although it is described as a neurodegenerative disease, due to expression of HTT in all types of cells, peripheral symptoms also occur. R6/1 and R6/2 mouse lines, which demonstrate many different phenotypical disturbances, are among the most commonly used HD animal models. Nevertheless, in this report, we underlined, for the first time, a previously undescribed R6/1 and R6/2 feature, hair dysmorphology. We observed changes in the general view of pelage, as well as specific changes in the shape of hair, assessed under electron microscope (deep cavity and hilly hair surface or concave and convex areas on the long hair axis with an appearance of the hair as flat). Hair diameter was significantly increased in both HD mouse models relative to control animals. Moreover, loosened contact between the scales and loosened scale texture were observed in R6/1 and R6/2. Thus, this study highlighted that the hair morphology might be a useful, noninvasive and simple marker of a widely used HD mouse models, R6/1 and R6/2 lines, particularly in testing effects of potential therapeutics or disease progression.

Enhanced cardiac hypoxic injury in atherogenic dyslipidaemia results from alterations in the energy metabolism pattern.

OBJECTIVE: Dyslipidaemia is a major risk factor for myocardial infarction that is known to correlate with atherosclerosis in the coronary arteries. We sought to clarify whether metabolic alterations induced by dyslipidaemia in cardiomyocytes collectively constitute an alternative pathway that escalates myocardial injury. METHODS: Dyslipidaemic apolipoprotein E and low-density lipoprotein receptor (ApoE/LDLR) double knockout (ApoE-/-/LDLR-/-) and wild-type C57BL/6 (WT) mice aged six months old were studied. Cardiac injury under reduced oxygen supply was evaluated by 5 min exposure to 5% oxygen in the breathing air under electrocardiogram (ECG) recording and with the assessment of troponin I release. To address the mechanisms LC/MS was used to analyse the cardiac proteome pattern or in vivo metabolism of stable isotope-labelled substrates and HPLC was applied to measure concentrations of cardiac high-energy phosphates. Furthermore, the effect of blocking fatty acid use with ranolazine on the substrate preference and cardiac hypoxic damage was studied in ApoE-/-/LDLR-/- mice. RESULTS: Hypoxia induced profound changes in ECG ST-segment and troponin I leakage in ApoE-/-/LDLR-/- mice but not in WT mice. The evaluation of the cardiac proteomic pattern revealed that ApoE-/-/LDLR-/- as compared with WT mice were characterised by coordinated increased expression of mitochondrial proteins, including enzymes of fatty acids' and branched-chain amino acids' oxidation, accompanied by decreased expression levels of glycolytic enzymes. These findings correlated with in vivo analysis, revealing a reduction in the entry of glucose and enhanced entry of leucine into the cardiac Krebs cycle, with the cardiac high-energy phosphates pool maintained. These changes were accompanied by the activation of molecular targets controlling mitochondrial metabolism. Ranolazine reversed the oxidative metabolic shift in ApoE-/-/LDLR-/- mice and reduced cardiac damage induced by hypoxia. CONCLUSIONS: We suggest a novel mechanism for myocardial injury in dyslipidaemia that is consequent to an increased reliance on oxidative metabolism in the heart. The alterations in the metabolic pattern that we identified constitute an adaptive mechanism that facilitates maintenance of metabolic equilibrium and cardiac function under normoxia. However, this adaptation could account for myocardial injury even in a mild reduction of oxygen supply.

The metabolism of ecto-5'-nucleotidase (CD73) inhibitor-α,ß-methylene adenosine diphosphate in BALB/c mice.

CD73 inhibitors are considered to be used in the therapies of melanomas, gliomas or breast cancer. However, little is known about their pharmacology and kinetics in mouse experimental models. Thus, this study is aimed to define a metabolic stability and elimination of the adenosine diphosphate (ADP) analog - α,ß-Methylene-ADP also known as AOPCP in BALB/c mice. The process starts with an intravenous injection of AOPCP, next blood and serum samples are collected. Urine samples are possessed by a bladder puncture. Mice aortas are dissected for the e5NT activity evaluation. In order to assess the AOPCP degradation, the incubation of AOPCP in mice blood and plasma is performed. The AOPCP concentration as well as the activity of e5NT were analyzed with the reverse phase-high pressure liquid chromatography (RP-HPLC). The study shows that after 60 minutes of the 20 mg/kg intravenous injection of AOPCP (body weight dose), the concentration of AOPCP in blood diminished rapidly from 38.6 ± 5.0 µM (measured 5 minutes after the injection) to 6.4 ± 1.4 µM. Interestingly, it is also noted that 60 minutes after the incubation of mice blood samples the AOPCP concentration decreases from 50 µM to 30.0 ± 0.3 µM. This study demonstrates a significant and quick decrease of AOPCP concentration in BALB/c mice blood after the intravenous injection and in isolated blood sample incubation. These findings emphasize the quick elimination of AOPCP as well as its instability and suggest that the AOPCP concentration have to be accurately and frequently monitored in all the studies that address its clinical application.