Metabolomic analysis of dietary-restriction-induced attenuation of sarcopenia in prematurely aging DNA repair-deficient mice

Background: Sarcopenia is characterized by loss of skeletal muscle mass and function, and is a major risk factor for disability and independence in the elderly. Effective medication is not available. Dietary restriction (DR) has been found to attenuate aging and aging-related diseases, including sarcopenia, but the mechanism of both DR and sarcopenia are incompletely understood. Methods: In this study, mice body weight, fore and all limb grip strength, and motor learning and coordination performance were first analysed to evaluate the DR effects on muscle functioning. Liquid chromatography–mass spectrometry (LC–MS) was utilized for the metabolomics study of the DR effects on sarcopenia in progeroid DNA repair-deficient Ercc1∆/− and Xpg−/− mice, to identify potential biomarkers for attenuation of sarcopenia. Results: Muscle mass was significantly (P &lt; 0.05) decreased (13–20%) by DR; however, the muscle quality was improved with retained fore limbs and all limbs grip strength in Ercc1∆/− and Xpg−/− mice. The LC–MS results revealed that metabolites and pathways related to oxidative-stress, that is, GSSG/GSH (P &lt; 0.01); inflammation, that is, 9-HODE, 11-HETE (P &lt; 0.05), PGE2, PGD2, and TXB2 (P &lt; 0.01); and muscle growth (PGF2α) (P &lt; 0.01) and regeneration stimulation (PGE2) (P &lt; 0.05) are significantly downregulated by DR. On the other hand, anti-inflammatory indicator and several related metabolites, that is, β-hydroxybutyrate (P &lt; 0.01), 14,15-DiHETE (P &lt; 0.0001), 8,9-EET, 12,13-DiHODE, and PGF1 (P &lt; 0.05); consumption of sources of energy (i.e., muscle and liver glycogen); and energy production pathways, that is, glycolysis (glucose, glucose-6-P, fructose-6-P) (P &lt; 0.01), tricarboxylic acid cycle (succinyl-CoA, malate) (P &lt; 0.001), and gluconeogenesis-related metabolite, alanine (P &lt; 0.01), are significantly upregulated by DR. The notably (P &lt; 0.01) down-modulated muscle growth (PGF2α) and regeneration (PGE2) stimulation metabolite and the increased consumption of glycogen in muscle and liver may be related to the significantly (P &lt; 0.01) lower body weight and muscle mass by DR. The downregulated oxidative stress, pro-inflammatory mediators, and upregulated anti-inflammatory metabolites resulted in a lower energy expenditure, which contributed to enhanced muscle quality together with upregulated energy production pathways by DR. The improved muscle quality may explain why grip strength is maintained and motor coordination and learning performance are improved by DR in Ercc1∆/− and Xpg−/− mice. Conclusions: This study provides fundamental supporting information on biomarkers and pathways related to the attenuation of sarcopenia, which might facilitate its diagnosis, prevention, and clinical therapy.</p

Metabolomic analysis of dietary-restriction-induced attenuation of sarcopenia in prematurely aging DNA repair-deficient mice

Background: Sarcopenia is characterized by loss of skeletal muscle mass and function, and is a major risk factor for disability and independence in the elderly. Effective medication is not available. Dietary restriction (DR) has been found to attenuate aging and aging-related diseases, including sarcopenia, but the mechanism of both DR and sarcopenia are incompletely understood. Methods: In this study, mice body weight, fore and all limb grip strength, and motor learning and coordination performance were first analysed to evaluate the DR effects on muscle functioning. Liquid chromatography–mass spectrometry (LC–MS) was utilized for the metabolomics study of the DR effects on sarcopenia in progeroid DNA repair-deficient Ercc1∆/− and Xpg−/− mice, to identify potential biomarkers for attenuation of sarcopenia. Results: Muscle mass was significantly (P &lt; 0.05) decreased (13–20%) by DR; however, the muscle quality was improved with retained fore limbs and all limbs grip strength in Ercc1∆/− and Xpg−/− mice. The LC–MS results revealed that metabolites and pathways related to oxidative-stress, that is, GSSG/GSH (P &lt; 0.01); inflammation, that is, 9-HODE, 11-HETE (P &lt; 0.05), PGE2, PGD2, and TXB2 (P &lt; 0.01); and muscle growth (PGF2α) (P &lt; 0.01) and regeneration stimulation (PGE2) (P &lt; 0.05) are significantly downregulated by DR. On the other hand, anti-inflammatory indicator and several related metabolites, that is, β-hydroxybutyrate (P &lt; 0.01), 14,15-DiHETE (P &lt; 0.0001), 8,9-EET, 12,13-DiHODE, and PGF1 (P &lt; 0.05); consumption of sources of energy (i.e., muscle and liver glycogen); and energy production pathways, that is, glycolysis (glucose, glucose-6-P, fructose-6-P) (P &lt; 0.01), tricarboxylic acid cycle (succinyl-CoA, malate) (P &lt; 0.001), and gluconeogenesis-related metabolite, alanine (P &lt; 0.01), are significantly upregulated by DR. The notably (P &lt; 0.01) down-modulated muscle growth (PGF2α) and regeneration (PGE2) stimulation metabolite and the increased consumption of glycogen in muscle and liver may be related to the significantly (P &lt; 0.01) lower body weight and muscle mass by DR. The downregulated oxidative stress, pro-inflammatory mediators, and upregulated anti-inflammatory metabolites resulted in a lower energy expenditure, which contributed to enhanced muscle quality together with upregulated energy production pathways by DR. The improved muscle quality may explain why grip strength is maintained and motor coordination and learning performance are improved by DR in Ercc1∆/− and Xpg−/− mice. Conclusions: This study provides fundamental supporting information on biomarkers and pathways related to the attenuation of sarcopenia, which might facilitate its diagnosis, prevention, and clinical therapy.</p

A sample preparation method for the simultaneous profiling of signaling lipids and polar metabolites in small quantities of muscle tissues from a mouse model for sarcopenia

The metabolic profiling of a wide range of chemical classes relevant to understanding sarcopenia under conditions in which sample availability is limited, e.g., from mouse models, small muscles, or muscle biopsies, is desired. Several existing metabolomics platforms that include diverse classes of signaling lipids, energy metabolites, and amino acids and amines would be informative for suspected biochemical pathways involved in sarcopenia. The sample limitation requires an optimized sample preparation method with minimal losses during isolation and handling and maximal accuracy and reproducibility. Here, two developed sample preparation methods, BuOH-MTBE-Water (BMW) and BuOH-MTBE-More-Water (BMMW), were evaluated and compared with previously reported methods, Bligh-Dyer (BD) and BuOH-MTBE-Citrate (BMC), for their suitability for these classes. The most optimal extraction was found to be the BMMW method, with the highest extraction recovery of 63% for the signaling lipids and 81% for polar metabolites, and an acceptable matrix effect (close to 1.0) for all metabolites of interest. The BMMW method was applied on muscle tissues as small as 5 mg (dry weight) from the well-characterized, prematurely aging, DNA repair-deficient Ercc1 Delta/-mouse mutant exhibiting multiple-morbidities, including sarcopenia. We successfully detected 109 lipids and 62 polar targeted metabolites. We further investigated whether fast muscle tissue isolation is necessary for mouse sarcopenia studies. A muscle isolation procedure involving 15 min at room temperature revealed a subset of metabolites to be unstable; hence, fast sample isolation is critical, especially for more oxidative muscles. Therefore, BMMW and fast muscle tissue isolation are recommended for future sarcopenia studies. This research provides a sensitive sample preparation method for the simultaneous extraction of non-polar and polar metabolites from limited amounts of muscle tissue, supplies a stable mouse muscle tissue collection method, and methodologically supports future metabolomic mechanistic studies of sarcopenia.Analytical BioScience

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled

PRMT3 inhibitor SGC707 reduces triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice

Protein arginine methyltransferase 3 (PRMT3) is a co-activator of liver X receptor capable of selectively modulating hepatic triglyceride synthesis. Here we investigated whether pharmacological PRMT3 inhibition can diminish the hepatic steatosis extent and lower plasma lipid levels and atherosclerosis susceptibility. Hereto, male hyperlipidemic low-density lipoprotein receptor knockout mice were fed an atherogenic Western-type diet and injected 3 times per week intraperitoneally with PRMT3 inhibitor SGC707 or solvent control. Three weeks into the study, SGC707-treated mice developed severe pruritus and scratching-associated skin lesions, leading to early study termination. SGC707-treated mice exhibited 50% lower liver triglyceride stores as well as 32% lower plasma triglyceride levels. Atherosclerotic lesions were virtually absent in all experimental mice. Plasma metabolite analysis revealed that levels of taurine-conjugated bile acids were ~ threefold increased (P < 0.001) in response to SGC707 treatment, which was paralleled by systemically higher bile acid receptor TGR5 signalling. In conclusion, we have shown that SGC707 treatment reduces hepatic steatosis and plasma triglyceride levels and induces pruritus in Western-type diet-fed LDL receptor knockout mice. These findings suggest that pharmacological PRMT3 inhibition can serve as therapeutic approach to treat non-alcoholic fatty liver disease and dyslipidemia/atherosclerosis, when unwanted effects on cholesterol and bile acid metabolism can be effectively tackled.Analytical BioScience

Noise and light pollution elicit endocrine responses in urban but not forest frogs

Urban areas are characterised by the presence of sensory pollutants, such as anthropogenic noise and artificial light at night (ALAN). Animals can quickly adapt to novel environmental conditions by adjusting their behaviour, which is proximately regulated by endocrine systems. While endocrine responses to sensory pollution have been widely reported, this has not often been linked to changes in behaviour, hampering the understanding of adaptiveness of endocrine responses. Our aim was, therefore, to investigate the effects of urbanisation, specifically urban noise and light pollution, on hormone levels in male urban and forest túngara frogs (Engystomops pustulosus), a species with reported population divergence in behaviour in response to urbanisation. We quantified testosterone and corticosterone release rates in the field and in the lab before and after exposure to urban noise and/or light. We show that urban and forest frogs differ in their endocrine phenotypes under field as well as lab conditions. Moreover, in urban frogs exposure to urban noise and light led, respectively, to an increase in testosterone and decrease in corticosterone, whereas in forest frogs sensory pollutants did not elicit any endocrine response. Our results show that urbanisation, specifically noise and light pollution, can modulate hormone levels in urban and forest populations differentially. The observed endocrine responses are consistent with the observed behavioural changes in urban frogs, providing a proximate explanation for the presumably adaptive behavioural changes in response to urbanisation

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Three-Phase Electroextraction: A New (Online) Sample Purification and Enrichment Method for Bioanalysis

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 μL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 μL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes

Profiling acidic metabolites by capillary electrophoresis-mass spectrometry in low numbers of mammalian cells using a novel chemical derivatization approach

The simultaneous analysis of a broad range of polar ionogenic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) can be challenging, as two different analytical methods are often required, that is, one for cations and one for anions. Even though CE-MS has shown to be an effective method for cationic metabolite profiling, the analysis of small anionic metabolites of ten results in relatively low sensitivity and poor repeatability. In this work, a novel derivatization strategy based on trimethyl-methaneaminophenacetyl bromide was developed to enable CE-MS analysis of carboxylicacid metabolites using normal CEpolarity (i.e.,cathode in the outlet) and detection by massspectrometry in positive ionization mode. Optimization of derivatization conditions was performed using a response surface methodology after which the optimized method (incubation time 50 min, temperature 90◦C, and pH10) was used for the analysis of carboxylicacidmetabolites in extracts from HepG2cells. For selected metabolites, detection limits were down to 8.2nM, and intraday relative standard deviation values for replicates (n=3) for peak areas were below 21.5%. Metabolites related to glycolysis, tricarboxylicacidcycle, and anaerobic respiration pathways were quantified in 250,000 cell lysates, and could still be detected in extracts from only 25,000 HepG2cell lysates (∼70 cell lysates injected)