Acute kidney injury (AKI) involves abrupt loss of kidney function driven in part by proximal tubule metabolic stress, yet the role of glycolytic regulation in tubular injury susceptibility remains unclear. Lactate dehydrogenase A (LDHA) is a key regulator of glycolytic flux and redox balance, but its function in proximal tubules during AKI is poorly defined. In this work, we use a cisplatin-induced AKI model to investigate the role of proximal tubule LDHA in regulating metabolic responses and injury severity. Proximal tubule-specific LDHA knockout mice (PEPCKCreLDHAΔ/Δ) and LDHAflox/flox controls were subjected to cisplatin-induced AKI. Untargeted metabolomics of kidney cortex and single-nucleus RNA sequencing (snRNA-seq) were performed to define metabolic and cell-specific transcriptional responses. Loss of proximal tubular LDHA exacerbated cisplatin-induced AKI, as evidenced by worsened kidney function and tubular injury, accompanied by increased expression of inflammatory markers following injury. The analysis also showed a distinct metabolic profile at baseline in LDHA-deficient kidneys, which became more pronounced after cisplatin exposure, with coordinated changes in purine and nucleotide metabolism, energy-related metabolites, and pathways linked to redox balance and mitochondrial function. snRNA-seq revealed intrinsic transcriptional changes within proximal tubule cells at baseline and after injury, reflecting cellular stress and metabolic remodeling without strong activation of classic inflammatory gene programs. Together, these findings identify proximal tubular LDHA as a key regulator of metabolic flexibility and injury tolerance in cisplatin-induced AKI, and suggest that disrupted coordination of glycolytic and nucleotide metabolism increases tubular vulnerability, highlighting metabolic regulation as a potential therapeutic target.NEW & NOTEWORTHY This study identifies proximal tubule lactate dehydrogenase A (LDHA) as a critical regulator of metabolic flexibility during cisplatin-induced acute kidney injury (AKI). Using a tissue-specific genetic approach and integrated multi-omics, we show that loss of LDHA worsens kidney injury and inflammatory responses while disrupting metabolic adaptation in proximal tubules. These findings highlight metabolic regulation within proximal tubules as a key determinant of injury tolerance and a potential therapeutic target in AKI.

IntroductionAlcohol use disorder (AUD) increases incidence of infections, organ damage, and cancers. Aberrant inflammation is likely a driver of these adverse outcomes. Indeed, chronic alcohol consumption (CAC) rewires macrophages/monocytes toward a hyper-inflammatory phenotype. Prior studies showed increased gut permeability and dysbiosis. Translocation of host- and microbial-derived metabolites could trigger the hyper-inflammatory responses generated by macrophages/monocytes. However, the exact changes in these metabolites remain poorly defined due to confounders that complicate clinical studies and the differences between human and rodent gut microbiomes.MethodsHere, we utilized a non-human primate model of ethanol self-administration to characterize alcohol-induced alterations in gut microbes and associated metabolomes. The microbiome was analyzed with 16s rRNA sequencing while a combination of GC-MS and LC-MS was used to assess changes in metabolites. Monocyte function was determined using flow cytometry.ResultsTwelve months of alcohol use led to a decrease in SCFA-producing bacteria and disruption of fatty acid and amino acid metabolites. Moreover, fecal metabolites obtained after 12 months of CAC heightened monocytes' inflammatory responses.DiscussionThese findings indicate that CAC-induced gut dysbiosis contributes to changes in fecal and circulating metabolites, which in turn can lead to monocyte dysregulation, possibly via innate immune training-like mechanisms.

Molecules in living systems are not random but are shaped by biological necessity. Mass spectrometry (MS) is a powerful tool for exploring these complex molecular landscapes. Molecular networking links metabolites by spectral similarity, but conventional methods leave many nodes disconnected. We introduce molecular community networking (MCN), which identifies natural molecular clusters and prunes them to keep the strongest links. The approach increases connectivity to about 95% of molecules and better captures structurally related compounds, including distinct ion forms and in-source fragmentation ions. MCN also improves the mapping of molecular space, helping distinguish true novel molecules from artifacts. Using MCN, we discovered dipeptide-conjugated bile acids associated with Bifidobacterium breve and proposed structures for previously unexplored N-acyl amides that interact with G protein-coupled receptors. We also built a global metabolome map from public GNPS/MassIVE data, covering about 8.4 million molecular features, creating a "roadmap" for molecular diversity.

Attention-deficit/hyperactivity disorder (ADHD) is prevalent among contact sports athletes, who may regularly incur repetitive head impacts. This study investigated the effects of acute head impacts on mitochondrial function by analyzing tricarboxylic acid (TCA) cycle metabolites and the potential modulatory role of ADHD. Fifty adult soccer players (ADHD n = 25; non-ADHD n = 25) participated, undergoing ten soccer headers using a controlled heading model. TCA metabolites were assessed at pre-heading baseline, and 2 and 24 h post-heading. Baseline analysis revealed elevated levels of TCA metabolites, including oxaloacetate, citrate, and isocitrate, in the ADHD group. Following head impacts, both groups exhibited significant decreases in these metabolites, yet the magnitude of decrease was more pronounced in the ADHD group. Pyruvate, alpha-ketoglutarate, and fumarate levels increased after headers in both groups. These findings suggest that ADHD is associated with elevated baseline metabolites initiating the TCA cycle, while acute head impacts induce mitochondrial dysfunction, regardless of ADHD.

Isoflavones are naturally occurring compounds found in a wide range of plants, but among commonly consumed foods are especially abundant in soybeans and foods derived from this legume. Much of the substantial amount of research conducted on soy protein and soy foods over the past 30 y is because of their isoflavone content. Research interest in isoflavones increased dramatically beginning in the early 1990s as evidence highlighted their possible role in the prevention of a wide range of cancers, including breast, prostate, and colon cancer. Recognition that isoflavones preferentially bind to estrogen receptor (ER)β in comparison with ERα provided a conceptual basis for classifying these diphenolic molecules as selective ER modulators (SERMs). Isoflavone research soon greatly expanded beyond cancer to include areas such as coronary artery disease, bone health, cognitive function, and vasomotor symptoms of menopause. Nevertheless, safety concerns about isoflavones, based primarily on the results of rodent studies and presumed estrogenic effects, also arose. However, recent work challenges the traditional view of the estrogenicity of isoflavones. Furthermore, safety concerns have largely been refuted by intervention and population studies. On the other hand, investigation of the proposed benefits of isoflavones has produced inconsistent data. The small sample size and short duration common to many intervention trials, combined with marked interindividual differences in isoflavone metabolism, likely contribute to the conflicting findings. Also, many different intervention products have been employed, which vary not only in the total amount, but also in the relative proportion of the 3 soybean isoflavones, and the form in which they are delivered (glycoside compared with aglycone). For those interested in exploring the proposed benefits of isoflavones, studies justify an intake recommendation of ∼50 mg/d, an amount provided by ∼2 servings of traditional Asian soy foods.

Background: Dietary interventions play a significant role in preventing and managing cardiometabolic diseases partly through their impact on the gut microbiome and circulating metabolites. Objectives: To assess the impact of an 8-week low-carbohydrate, high-protein (LC/HP) diet on gut microbiome composition, function, and serum metabolome in individuals with spinal cord injury (SCI). Methods: Twenty-four adults with chronic SCI were randomized into an LC/HP diet or a control group for 8 weeks. Stool and fasting serum samples were collected at baseline and week 8. The gut microbiome composition and metabolic potential were determined using metagenomic sequencing, while serum metabolome was assessed through untargeted liquid chromatography-tandem mass spectrometry. Statistical analyses focused on diet and time interaction effects, using R (version 4.1.0). Results: A trend for increased alpha diversity (Gini-Simpson, P = .09) in the diet group indicated a more evenly distributed microbial community. Compared to the control group, several microbiome species (e.g., Fusicatenibacter saccharivorans, Eubacterium siraeum) that are implicated with better intestinal health and reduced inflammation increased, while other species (e.g., Hungatella hathewayi, Clostridium symbiosum) that are associated with colorectal cancer risk decreased in the diet group. Microbial metabolic pathways related to amino acid and purine nucleotides were altered. Increased tryptophan betaine and decreased 8-hydroxy-deoxyguanosine were observed in the serum in the diet group (Pinteraction < .05), indicating compliance and reduced oxidative stress, respectively. Conclusion: Adopting an LC/HP diet resulted in favorable gut microbiome and metabolome adaptations that may reduce the risk for cardiometabolic disease and colorectal cancer in individuals with SCI.

INTRODUCTION: Metabolomics can comprehensively review biochemical changes driving lung disease by identifying and quantifying various metabolites in bronchoalveolar lavage fluids (BALF) for mechanistic insights. Shared lung anatomy and physiology in ferrets enable the close approximation of COPD and increased susceptibility to infections by pathogens associated with clinical exacerbations. Previously, we showed that smoking increased the H1N1 viral load in ferrets. Here, we profile BALF metabolites in control and smoke-exposed ferrets with and without viral infection representative of exacerbation. METHODS: Wild type, age, and sex-matched ferrets were exposed to cigarette smoke (CS) for one month (1R6F), followed by a sublethal (intranasal) infection with 106 H1N1 (A/California/07/2009). We collected BALF four days after infection, extracted metabolites using the methanol-formic acid method, and analyzed them in an LC-MS/MS-based untargeted metabolomic platform with positive and negative IonSpray modes. We identified peak area and retention time using MS-Dial and analyzed differential metabolites and associated pathways using MetaboAnalyst. RESULTS: Partial Least-Squares Discriminant Analysis showed a distinct separation of metabolite profiles based on the treatments. Compared to air control, smoke, viral infection, and smoke+virus significantly altered 16, 26, and 31 metabolites. Of the 31, 13 metabolites were commonly driven by both smoke and virus and were mostly nucleosides/nucleotides, indicating a pivotal role of altered purine and pyrimidine metabolism. Irrespective of whether the animals were exposed to CS or not, H1N1 infection significantly altered metabolomic profiles; however, with CS exposure, infected BALF was characterized by increased levels of certain amino acids. CONCLUSIONS: Beyond reducing antiviral immunity that underlies the increased viral burden among smokers and COPD patients, smoking alone causes inflammatory metabolic changes that are synergistically exaggerated by viral infections. These data point to distinct and additive pathogenic mechanisms between smoking and viruses during COPD exacerbation that deserve additional research attention. These data also explain how efficacious drugs that target stable COPD reduce the frequency and severity of future exacerbations.

BACKGROUND & AIMS:Duodenal adenomas have malignant potential, yet the drivers of duodenal tumorigenesis remain unclear. Duodenal adenomas robustly develop in villin- Toll-like receptor 4 (TLR4) mice, a transgenic mouse model of increased innate immune signaling in the intestinal epithelium. Here, we sought to test the contributions of the microbiota and bile acids to duodenal adenoma development. METHODS:Duodenal tissue was analyzed for proliferation rate and histology in villin-TLR4 vs wild-type mice. Mice were rederived into germ-free conditions and administered a diet containing the bile acid sequestering resin cholestyramine or treated with the NADPH oxidase inhibitor apocynin. Chemokine expression and myeloid cell recruitment were measured. Findings from mouse studies were corroborated by RNA sequencing and tissue microarray analyses of human duodenal adenomas. RESULTS:Constitutive activation of epithelial TLR signaling in the duodenum led to adenomas with an intestinal phenotype. Non-adenomatous duodenal tissue showed increased expression of Cxcl1 and Cxcl2 by intestinal epithelial cells and recruitment of S100A8+ and myeloperoxidase+ myeloid cells. Re-deriving villin-TLR4 mice in germ-free conditions or feeding them a cholestyramine-supplemented diet prevented tumor initiation, epithelial expression of CXCR2 ligands, and myeloid cell recruitment. Apocynin supplementation slowed tumor progression without affecting chemokine expression or myeloid cell recruitment. In humans, duodenal adenomas had enriched neutrophil activation pathways, increased chemokine expression, and infiltration of S100A8+ and myeloperoxidase+ myeloid cells. CONCLUSIONS:Bile acids and the microbiota are necessary for duodenal adenoma development and are potentially modifiable risk factors in humans at risk of duodenal adenomas. The recruitment of myeloid cells may promote tumor progression via the release of reactive oxygen species.

Key PointsLactate dehydrogenase A deletion alters macrophage function.Lactate dehydrogenase A could serve as a potential therapeutic target in AKI.BackgroundIn AKI, macrophages play a major role in regulating inflammation. Classically activated macrophages (M1) undergo drastic metabolic reprogramming during their differentiation and upregulate the aerobic glycolysis pathway to fulfill their proinflammatory functions. NAD+ regeneration is crucial for the maintenance of glycolysis, and the most direct pathway by which this occurs is through the fermentation of pyruvate to lactate, catalyzed by lactate dehydrogenase A (LDHA). Our previous study determined that LDHA is predominantly expressed in the proximal segments of the nephron in the mouse kidney and increases with hypoxia. This study investigates the potential of LDHA as a therapeutic target for inflammation by exploring its role in macrophage function in vitro.MethodsBone marrow-derived macrophages (BMDMs) were isolated from myeloid-specific LDHA knockout mice derived from crossbreeding LysM-Cre transgenic mice and LDHA floxed mice. RNA sequencing and LC-MS/MS metabolomics analyses were used in this study to determine the effect of LDHA deletion on BMDMs after stimulation with IFN-gamma.ResultsLDHA deletion in IFN-gamma BMDMs resulted in a significant alteration of the macrophage activation and functional pathways and change in glycolytic, cytokine, and chemokine gene expression. Metabolite concentrations associated with proinflammatory macrophage profiles were diminished, whereas anti-inflammatory-associated ones were increased in LDHA knockout BMDMs. Glutamate and amino sugar metabolic pathways were significantly affected by the LDHA deletion. A combined multiomics analysis highlighted changes in Rap1 signaling, cytokine-cytokine receptor interaction, focal adhesion, and mitogen-activated protein kinase signaling metabolism pathways.ConclusionsDeletion of LDHA in macrophages results in a notable reduction in the proinflammatory profile and concurrent upregulation of anti-inflammatory pathways. These findings suggest that LDHA could serve as a promising therapeutic target for inflammation, a key contributor to the pathogenesis of AKI.

AIM:Sirtuin 5 (SIRT5), a mitochondrial NAD+-dependent deacylase, regulates fundamental cellular pathways, including energy substrate metabolism. The current study is designed to better elucidate the role of SIRT5 in the development of heart failure (HF). METHODS:Mice with cardiomyocyte-specific deletion (cSirt5-/-) or overexpression (cSirt5-Tg) of SIRT5 were generated and subjected to chronic pressure overload by transverse aortic constriction (TAC) or Sham surgery. Cardiac structure and function were assessed by echocardiography, isolated heart perfusions, and histology. MS-based metabolomics and bulk RNA sequencing were used to explore metabolic and molecular signatures. RESULTS:cSirt5-Tg mice had similar cardiac structure and function compared to control mice, whereas cSirt5-/- mice displayed exacerbated cardiac dilation and dysfunction following TAC, measured both in vivo by echocardiography and ex vivo in isolated heart perfusions. Metabolomics revealed accumulation of inosine and hypoxanthine, and depletion of adenosine, adenine, AMP, and ADP in cSirt5-/- hearts and following TAC, indicating dysregulation of purine metabolism. RNA-sequencing uncovered upregulation of purine-nucleoside phosphorylase and 5' nucleotidase, and downregulation of adenosine kinase (ADK) in cSirt5-/- hearts following TAC, indicating dysregulation at the interface of adenosine nucleotide salvage and purine degradation in the absence of SIRT5. Analyses of left ventricular tissue of patients with HF revealed reduced SIRT5 expression correlating with reduced ADK expression. CONCLUSION:Loss of SIRT5 in cardiomyocytes aggravates cardiac remodeling and dysfunction in response to chronic pressure overload, involving ATP precursor depletion due to transcriptional dysregulation of cardiac purine metabolism.

Dietary load and composition are known contributors that accelerate cyst growth in polycystic kidney disease (PKD). High protein intake, which increases amino acid burden in the kidneys, is one such factor. Despite identical protein load, a plant-based wheat-gluten (WG) diet was recently reported to blunt the inflammatory response of animal-based casein diet in a hypertensive model. Considering the importance of pro-inflammatory signals on cystogenesis in PKD, we therefore sought to determine whether a WG compared to casein diet would decelerate cyst progression. Tamoxifen-inducible, global Pkd1 knockout mice were fed either a low casein (6%), high casein (60%), or high wheat-gluten (60%) protein diet for 6 wk. In a separate cohort, mice were gavaged daily with vehicle, lysine, or glutamine for 4 wk while maintained on a normal protein (18%) diet. Tissues were used for histology, flow cytometry, mitochondrial function, metabolomics, and various biochemical assays. WG-fed mice had better kidney function and reduced kidney macrophage percentages, proinflammatory cytokine expression, and cyst growth compared to casein-fed mice. Protein source did not alter kidney mitochondria function. Supplementation with lysine, the highest amino acid in casein versus WG diet, increased kidney cyst growth, acid production, and metabolic disarray. This did not occur with glutamine supplementation, the highest amino acid in WG versus casein diet, despite increased glomerular filtration rate with both amino acids. Neither supplementation mounted an inflammatory response. A plant-based, low-lysine diet slows disease burden in a murine model of PKD. This easily modifiable diet may be a beneficial intervention for PKD patients.

Emerging evidence indicates that the health‐beneficial effects of ingested food components depend on in part, their upper and lower gastrointestinal uptake and metabolism and the gut microbial composition of the host. Several dietary components, such as polyphenols, are poorly absorbed in the upper intestinal compartments and extensively metabolized by the colonic microbiota resulting in the production of an array of metabolites. These microbiota‐mediated products possess specific solubility, reactivity, bioavailability, and biological activities. However, identifying and characterizing a wide range of metabolites is challenging due to the high chemical diversity of dietary components and interindividual variability of the gut microbiota. It is, therefore, critical to design an animal model that effectively mimics human microbial metabolism and use multidisciplinary omics approaches such as metabolomics to detect and identify a wide range of metabolites. Here, we provide the current state of knowledge of major diet‐derived gut microbial metabolites and their potential biological activities.

PURPOSE:Trimethylamine-N-oxide (TMAO) is a gut-derived metabolite associated with cardiovascular disease (CVD). In preclinical and observational studies, resveratrol and exercise training have been suggested as potential strategies to reduce the systemic levels of TMAO. However, evidence from experimental studies in humans remains unknown. This project examined the dose-dependent effects of a combined resveratrol intervention with exercise training on circulating TMAO and other related metabolite signatures in older adults with high CVD risk. METHODS:Forty-one older adults [mean (±SD) age of 72.1 (6.8) years] participated in a 12-week supervised center-based, multi-component exercise training intervention [2×/week; 80 min/session] and were randomized to one of two resveratrol dosages [Low: 500 vs. High:1000 mg/day] or a cellulose-based placebo. Serum/plasma were collected at baseline and post-intervention and evaluated for TMAO and associated analytes. RESULTS:After the 12-week intervention, TMAO concentration increased over time, regardless of treatment [mean (±SD) Placebo: 11262 (±3970); Low:13252 (±1193); High: 12661(±3359) AUC; p = 0.04]. Each resveratrol dose produced different changes in metabolite signatures. Low dose resveratrol upregulated metabolites associated with bile acids biosynthesis (i.e., glycochenodeoxycholic acid, glycoursodeoxycholic acid, and glycocholic acid). High dose resveratrol modulated metabolites enriched for glycolysis, and pyruvate, propanoate, β-alanine, and tryptophan metabolism. Different communities tightly correlated to TMAO and resveratrol metabolites were associated with the lipid and vascular inflammatory clinical markers [|r| > 0.4, p < 0.05]. CONCLUSION:These findings suggest a distinct dose-dependent adaptation response to resveratrol supplementation on circulating metabolite signatures but not on TMAO among high-risk CVD older adults when combined with an exercise training intervention.

Accumulating evidence has revealed that alterations in the gut microbiome following spinal cord injury (SCI) exhibit similarities to those observed in metabolic syndrome. Considering the causal role of gut dysbiosis in metabolic syndrome development, SCI-induced gut dysbiosis may be a previously unidentified contributor to the increased risk of cardiometabolic diseases, which has garnered attention. With a cross-sectional design, we evaluated the correlation between gut microbiome composition and functional potential with indicators of metabolic health among 46 individuals with chronic SCI. Gut microbiome communities were profiled using next-generation sequencing techniques. Indices of metabolic health, including fasting lipid profile, glucose tolerance, insulin resistance, and inflammatory markers, were assessed through fasting blood tests and an oral glucose tolerance test. We used multivariate statistical techniques (i.e., regularized canonical correlation analysis) to identify correlations between gut bacterial communities, functional pathways, and metabolic health indicators. Our findings spotlight bacterial species and functional pathways associated with complex carbohydrate degradation and maintenance of gut barrier integrity as potential contributors to improved metabolic health. Conversely, those correlated with detrimental microbial metabolites and gut inflammatory pathways demonstrated associations with poorer metabolic health outcomes. This cross-sectional investigation represents a pivotal initial step toward comprehending the intricate interplay between the gut microbiome and metabolic health in SCI. Furthermore, our results identified potential targets for future research endeavors to elucidate the role of the gut microbiome in metabolic syndrome in this population.NEW & NOTEWORTHY Spinal cord injury (SCI) is accompanied by gut dysbiosis and the impact of this on the development of metabolic syndrome in this population remains to be investigated. Our study used next-generation sequencing and multivariate statistical analyses to explore the correlations between gut microbiome composition, function, and metabolic health indices in individuals with chronic SCI. Our results point to potential gut microbial species and functional pathways that may be implicated in the development of metabolic syndrome.

Rapamycin slows cystogenesis in murine models of polycystic kidney disease (PKD) but failed in clinical trials, potentially due to insufficient drug dosing. To improve drug efficiency without increasing dose, kidney-specific drug delivery may be used. Mesoscale nanoparticles (MNP) selectively target the proximal tubules in rodents. We explored whether MNPs can target cystic kidney tubules and whether rapamycin-encapsulated-MNPs (RapaMNPs) can slow cyst growth in Pkd1 knockout (KO) mice. MNP was intravenously administered in adult Pkd1KO mice. Serum and organs were harvested after 8, 24, 48 or 72 h to measure MNP localization, mTOR levels, and rapamycin concentration. Pkd1KO mice were then injected bi-weekly for 6 weeks with RapaMNP, rapamycin, or vehicle to determine drug efficacy on kidney cyst growth. Single MNP injections lead to kidney-preferential accumulation over other organs, specifically in tubules and cysts. Likewise, one RapaMNP injection resulted in higher drug delivery to the kidney compared to the liver, and displayed sustained mTOR inhibition. Bi-weekly injections with RapaMNP, rapamycin or vehicle for 6 weeks resulted in inconsistent mTOR inhibition and little change in cyst index, however. MNPs serve as an effective short-term, kidney-specific delivery system, but long-term RapaMNP failed to slow cyst progression in Pkd1KO mice.

Purpose:The purpose of this study was to explore the effects of a PGF2α analog, latanoprost, and its preservative, benzalkonium chloride (BAK), on the cell viability and lipidomic expression of immortalized human meibomian gland epithelial cells (HMGECs). Methods:Differentiated HMGECs were exposed to latanoprost (0.05 to 50 µg/ml), BAK (0.2 to 200 µg/ml), or combined latanoprost-BAK (0.05-0.2 to 50-200 µg/ml). EP- and FP-type receptors, the cognate receptors of PGE2 and PGF2α, were inhibited, thereby sparing and isolating the function of each receptor to one condition. Cell viability was assessed by ATP quantitation, and lipid extracts were analyzed by ESI-MSMSALL with a Triple TOF 5600 Mass Spectrometer (SCIEX, Framingham, MA) using SCIEX LipidView 1.3. Results:Latanoprost and BAK were found to be lethal to HMGECs at the highest concentrations (p < 0.001 for both). The cytotoxicity of latanoprost was mediated through FP- and EP-independent mechanisms. Both latanoprost and BAK significantly modulated the lipidomic expression of several cholesteryl esters (8% and 30%, respectively) and triacylglycerols (10% and 12%, respectively). The combined latanoprost-BAK agent appeared to be no more toxic and to only negligibly alter the lipid profile relative to its individual components. Conclusions:The use of latanoprost and BAK in glaucoma may alter the viability of the meibomian glands and their lipid expression in vivo. Sublethal concentrations of BAK appear to modulate meibum lipid expression, particularly in relation to sterol biosynthesis. Non-preserved latanoprost had less cytotoxicity at lower doses and fewer lipidomic effects compared to BAK, further strengthening the argument in favor of BAK-free pharmaceutical preparations.

The extracellular cellular matrix (ECM) maintains tissue structure and regulates signaling functions by continuous degradation and remodeling. Inflammation or other disease conditions activate proteases including matrix metalloproteinases (MMPs) that degrade ECM proteins and in particular generate fragments of collagen and elastin, some of which are biologically active ECM peptides or matrikines. Stepwise degradation of collagen by MMP 8, 9 and prolyl endopeptidase release the matrikine proline-glycine-proline (PGP) and its product acetyl-PGP (AcPGP). These peptides are considered as potential biomarkers and therapeutic targets for many disease conditions such as chronic lung disease, heart disease, and cancer. However, there is no published, validated method for the measurement of PGP and AcPGP in plasma and therefore, we developed a sensitive, selective and reliable, isotope dilution LC-multiple reaction monitoring MS method for their determination in human plasma. The chromatographic separation of PGP and AcPGP was achieved in 3 min using Jupiter column with a gradient consisting of acidified acetonitrile and water at a flow rate of 0.5 ml/min. The limit of detection (LOD) for PGP and AcPGP was 0.01 ng/ml and the limit of quantification (LOQ) was 0.05 ng/ml and 0.1 ng/ml, respectively. Precision and accuracy values for all analytes were within 20 % except for the lowest QC of 0.01 ng/ml. The mean extraction recoveries of these analytes were > 90 % using a Phenomenex Phree cartridge and the matrix effect was < 15 % for all the QCs for PGP and AcPGP except the lowest QC. The stability of PGP and AcPGP was > 90 % in several tested conditions including autosampler use, storage at −80 °C, and after 6 times freeze–thaw cycles. Using this method, we successfully extracted and determined PGP levels in human plasma from healthy and COPD subjects. Therefore, this method is suitable for quantification of these peptides in the clinical setting.

Obesity caused by genetic and environmental factors can lead to compromised skeletal muscle function. Time-restricted feeding (TRF) has been shown to prevent muscle function decline from obesogenic challenges; however, its mechanism remains unclear. Here we demonstrate that TRF upregulates genes involved in glycine production (Sardh and CG5955) and utilization (Gnmt), while Dgat2, involved in triglyceride synthesis is downregulated in Drosophila models of diet- and genetic-induced obesity. Muscle-specific knockdown of Gnmt, Sardh, and CG5955 lead to muscle dysfunction, ectopic lipid accumulation, and loss of TRF-mediated benefits, while knockdown of Dgat2 retains muscle function during aging and reduces ectopic lipid accumulation. Further analyses demonstrate that TRF upregulates the purine cycle in a diet-induced obesity model and AMPK signaling-associated pathways in a genetic-induced obesity model. Overall, our data suggest that TRF improves muscle function through modulations of common and distinct pathways under different obesogenic challenges and provides potential targets for obesity treatments. Time-restricted feeding (TRF) can prevent muscle function decline from obesogenic challenges. Here, the authors reveal that TRF improves muscle function through modulations of common and distinct pathways in diet- and genetic-induced obesity models.