Metabolites

17 pages, 3203 KB

Open AccessProtocol

Optimizing the Seahorse XF Mito Stress Test Workflow and Troubleshooting Notes: A Stepwise Protocol for HUVECs

by Jingyi Wang, Yue Jiao, Jingzhe Li, Yanyan Ma, Changzhen Liu and Jing Yang

Metabolites 2026, 16(2), 99; https://doi.org/10.3390/metabo16020099 - 28 Jan 2026

This protocol details an optimized step-by-step procedure for performing the Seahorse XF Cell Mito Stress Test on human umbilical vein endothelial cells (HUVECs) using the Agilent Seahorse XF Pro Analyzer. Designed to address practical challenges often overlooked in standard manuals, the method preserves [...] Read more.

This protocol details an optimized step-by-step procedure for performing the Seahorse XF Cell Mito Stress Test on human umbilical vein endothelial cells (HUVECs) using the Agilent Seahorse XF Pro Analyzer. Designed to address practical challenges often overlooked in standard manuals, the method preserves the native adherent state of HUVECs—a key in vitro model in vascular aging (VA) research—enabling real-time, label-free measurement of mitochondrial respiration and glycolytic function without cell detachment. The workflow is presented chronologically, covering instrument preparation, cell seeding, compound loading, assay execution, and post-assay normalization, with integrated notes and troubleshooting tips refined through hands-on experience based on the official manuals. This protocol aims to set up a detailed, rearranged standard workflow to improve experimental efficiency, reduce operator error, and support reproducible and well-organized metabolic profiling of HUVECs in aging and cardiovascular studies. Full article

(This article belongs to the Section Cell Metabolism)

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26 pages, 769 KB

Open AccessSystematic Review

Myoclonus in Pediatric Metabolic Diseases: Clinical Spectrum, Mechanisms, and Treatable Causes—A Systematic Review

by Elżbieta Majewska, Zofia Zdort, Aleksandra Ochocka and Justyna Paprocka

Metabolites 2026, 16(2), 98; https://doi.org/10.3390/metabo16020098 - 28 Jan 2026

Abstract

Background: Myoclonus, a sudden brief shock-like involuntary movement, represents a common yet under-recognized manifestation across many inherited metabolic disorders. Although its occurrence has been reported in case series and small cohorts, the overall spectrum, pathophysiological mechanisms, and therapeutic relevance of metabolic myoclonus [...] Read more.

Background: Myoclonus, a sudden brief shock-like involuntary movement, represents a common yet under-recognized manifestation across many inherited metabolic disorders. Although its occurrence has been reported in case series and small cohorts, the overall spectrum, pathophysiological mechanisms, and therapeutic relevance of metabolic myoclonus have not been systematically summarized. Methods: A systematic search of PubMed was conducted for English-language publications from 2014 to 2025 using predefined MeSH terms related to myoclonus, movement disorders, and inborn errors of metabolism. Titles and abstracts were screened independently by three reviewers. After removal of duplicates, 27 articles were included, complemented by 65 additional references addressing individual disorders. Data were organized according to the International Classification of Inherited Metabolic Disorders (ICIMD). Results: Myoclonus was documented across six ICIMD categories, including intermediary metabolism, mitochondrial energy metabolism, lipid metabolism, disorders of complex molecules and organelles, cofactor and mineral metabolism, and metabolic cell signaling disorders. Clinical presentation ranged from isolated jerks to progressive myoclonic epilepsies. Several conditions—such as GLUT1 deficiency, cerebrotendinous xanthomatosis, and folate receptor α deficiency—are treatable through dietary or pharmacological interventions. Conclusions: Recognition of myoclonus as a presenting feature of inherited errors of metabolism (IEMs) is critical for timely diagnosis and treatment. Metabolic screening should be considered in all unexplained cases of myoclonus, particularly when accompanied by developmental delay or systemic abnormalities. Full article

(This article belongs to the Section Cell Metabolism)

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16 pages, 13139 KB

Open AccessArticle

Exploratory Analysis of Gut Microbiome and Metabolic Profile Changes Following Lenvatinib and Anti-PD-1 Combination Therapy in Liver Cancer

by Rui Zhong, Zhikun Lin, Binghui Jin, Xiaolin Wang, Hua Mu, Jinlong Hu, Qi Li, Peng Dou, Xinyu Liu, Chunxiu Hu, Guowang Xu and Guang Tan

Metabolites 2026, 16(2), 97; https://doi.org/10.3390/metabo16020097 - 28 Jan 2026

Abstract

Background/Objectives: Lenvatinib combined with anti-PD-1 therapy has shown promise in the treatment of hepatocellular carcinoma (HCC). The study evaluates changes in gut microbiota (GM) and metabolites during HCC treatment with lenvatinib combined with anti-PD-1. Methods: An HCC mouse model was established [...] Read more.

Background/Objectives: Lenvatinib combined with anti-PD-1 therapy has shown promise in the treatment of hepatocellular carcinoma (HCC). The study evaluates changes in gut microbiota (GM) and metabolites during HCC treatment with lenvatinib combined with anti-PD-1. Methods: An HCC mouse model was established via diethylnitrosamine (DEN) injection, and the mice were then treated with lenvatinib, anti-PD-1, or their combination. GM composition and structural changes were assessed by 16S rDNA sequencing, and metabolite abundance by liquid chromatography–mass spectrometry (LC–MS). Results: Significant alterations in GM and metabolites were observed in the HCC group compared to the control group, and compared with the HCC group, both monotherapy and combination therapy resulted in varying degrees of GM and metabolites rebalancing. Specifically, compared to the HCC group, lenvatinib combined with anti-PD-1 therapy decreased the abundance of GM, including p_Patescibacteria, g_Lactobacillus, g_Clostridium_sensu_stricto_1, g_Eubacterium_siraeum_group, and g_Desulfovibrio, while the abundance of g_Prevotella_7 increased. Metabolite changes included increased 4-pyridoxic acid, deoxycholic acid, and taurochenodesoxycholic acid, and decreased myristic acid, oleic acid, riboflavin, and uric acid. Conclusions: HCC induces substantial alterations in the GM and metabolic profile of mice. Lenvatinib combined with anti-PD-1 treatment partially modulates these dysregulations. The relevant GM and metabolites may be associated with the efficacy of combined therapy and could serve as potential markers for further investigation. Full article

(This article belongs to the Section Endocrinology and Clinical Metabolic Research)

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15 pages, 3084 KB

Open AccessArticle

Widely Targeted Liver Metabolomics Reveals Potential Biomarkers in Mice with Drug-Induced Liver Injury

by Jiangning Peng, Tingting Zhao, Xuehong Zhang, Hong Wang, Hui Li and Yan Liang

Metabolites 2026, 16(2), 96; https://doi.org/10.3390/metabo16020096 - 28 Jan 2026

Abstract

Background: Drug-induced liver injury (DILI), a major type of adverse drug reaction, has become one of the leading causes of acute liver injury and liver failure worldwide. Its clinical significance lies not only in acute hepatocyte necrosis and functional failure but also in [...] Read more.

Background: Drug-induced liver injury (DILI), a major type of adverse drug reaction, has become one of the leading causes of acute liver injury and liver failure worldwide. Its clinical significance lies not only in acute hepatocyte necrosis and functional failure but also in its role as a key initiating factor for liver cancer progression. Therefore, early diagnosis of DILI is of great importance. Methods: This study employed ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) to perform widely targeted metabolomics analysis on acetaminophen (APAP)-induced liver injury mice and healthy mice. Results: UPLC-QTRAP-MS/MS identified 41 differentially expressed metabolites primarily involved in glycerophospholipid metabolism, arginine and proline metabolism, primary bile acid biosynthesis, and glutathione metabolism pathways. The significant elevation of serum and hepatic alanine aminotransferase (ALT) and aspartate aminotransferase (AST) confirmed the successful establishment of the drug-induced liver injury (DILI) model. ROC curve analysis indicated 11 metabolites with AUC values exceeding 0.90 as potential biomarkers, including (R)-2-Hydroxybutyric acid, Glu-Gln, γ-Glu-Gln, 2-Methyllactic acid, L-Serine, Hyodeoxycholic acid, 3-Epideoxycholic acid, and Glycochenodeoxycholic acid 7-sulfate. Conclusions: We propose that these differential metabolites may serve as candidate biomarkers for DILI. Our findings provide a novel metabolomic signature derived directly from the injured tissue and offer a theoretical foundation for further research into early diagnosis of drug-induced liver injury. Full article

(This article belongs to the Section Endocrinology and Clinical Metabolic Research)

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22 pages, 1738 KB

Open AccessArticle

Tissue-Specific Multi-Omics Integration Demonstrates Molecular Signatures Connecting Obesity to Immune Vulnerability

by Ozge Onluturk Aydogan, Aytac Dursun Oksuzoglu and Beste Turanli

Metabolites 2026, 16(2), 95; https://doi.org/10.3390/metabo16020095 - 27 Jan 2026

Abstract

Background: Adipose tissue surrounds organs and tissues in the body and can alter their function. It could secrete diverse biological molecules, including lipids, cytokines, hormones, and metabolites. In light of all this information, obesity can influence many tissues and organs in the body, [...] Read more.

Background: Adipose tissue surrounds organs and tissues in the body and can alter their function. It could secrete diverse biological molecules, including lipids, cytokines, hormones, and metabolites. In light of all this information, obesity can influence many tissues and organs in the body, and this situation makes obesity a central contributor to multiple disorders. It is very important to investigate the crosstalk between tissues and organs in the body to clarify the key mechanisms of obesity. Methods: In this study, we analyzed the gene expression profiles of the liver, skeletal muscle, blood, visceral, and subcutaneous adipose tissue. Differentially expressed genes (DEGs) were identified for each tissue, and functional enrichment and protein–protein interaction network analyses were performed on genes commonly identified across tissues. Priority candidate genes were identified using network-based centrality measures, and potential molecular intersection points were explored through host-pathogen interaction network analysis. This study provides an integrative framework for characterizing inter-tissue molecular patterns associated with obesity at the network level. Results: The muscle, subcutaneous adipose tissue, and blood have the highest number of DEGs. The subcutaneous adipose tissue and blood stand out due to the number of DEGs they possess, although liver and visceral adipose tissue have lower amounts. Cancer ranks first in terms of diseases associated with obesity, and this association is accompanied by leukemia, lymphoma, and gastric cancer. RPL15 and RBM39 are the top genes in both degree and betweenness metrics. The host–pathogen interaction network consists of 13 unique-host proteins, 54 unique-pathogen proteins, and 27 unique-pathogen organisms, and the Influenza A virus had the highest interaction. There were a small number of common metabolites in all tissues: 2-Oxoglutarate, Adenosine, Succinate, and D-mannose. Conclusions: In this study, we aimed to identify candidate molecules for obesity using an integrative approach, examining the gene profiles of different organs and tissues. The findings of this study suggest a possible link between obesity and immune-related biological processes. The network obtained from the host-pathogen interaction analysis, and especially the pathways associated with viral infections that stand out in the functional enrichment analysis, may overlap with molecular signatures linked to obesity. Furthermore, the co-occurrence of cytokine signaling, insulin, and glucose metabolism pathways in the enrichment results indicates that the response of cells to insulin may be affected in obese individuals, suggesting a potential interaction between immune and metabolic processes; however, further experimental validation is needed to reveal the direct functional effects of these relationships. Full article

(This article belongs to the Special Issue Exploring Pathological Mechanisms in Obesity, Diabetes, and Metabolic Syndrome: 2nd Edition)

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22 pages, 1436 KB

Open AccessArticle

Effects of Leucine Supplementation on Athletic Performance, Central Fatigue, and Serum Metabolism in Endurance Athletes: A Randomized Controlled Trial and Targeted Metabolomics Study

by Tieying Li, Wei Xu, Jun Chen, Zhaobo Kan, Xuemei Sui, Zhiguang Zhao and Qirong Wang

Metabolites 2026, 16(2), 94; https://doi.org/10.3390/metabo16020094 - 27 Jan 2026

Abstract

Objectives: We aimed to investigate the effects of 6 weeks of leucine supplementation on athletic performance, central fatigue, and serum metabolome in endurance athletes, and to provide valuable insights into nutritional strategies for endurance athletes. Methods: Twenty cross-country skiers were recruited [...] Read more.

Objectives: We aimed to investigate the effects of 6 weeks of leucine supplementation on athletic performance, central fatigue, and serum metabolome in endurance athletes, and to provide valuable insights into nutritional strategies for endurance athletes. Methods: Twenty cross-country skiers were recruited and randomized into 2 groups: the placebo (PLA) group and the leucine (LEU) group. Subjects were given leucine (8.5 g) + sucrose (14 g) or only sucrose (14 g) supplements twice each day from Monday to Saturday for 6 weeks. Test parameters include body composition, aerobic capacity, isokinetic muscle strength, blood biochemistry, and targeted metabolomics. Results: After intervention, compared to the PLA group (1) the ankle muscle strength (p = 0.01), VO₂max (p = 0.01) and valine in serum (p = 0.03) were increased in the LEU group. (2) Targeted metabolomics results showed that the differential metabolites were enriched in the branched chain amino acids (BCAAs) biosynthesis and degradation. (3) The LEU group had a significant increase in α-ketoisovaleric acid (p = 0.03), which can reduce the continuous decomposition of BCAAs. Conclusions: In conclusion, a six-week intervention of leucine supplementation significantly enhanced ankle muscle strength in endurance athletes, likely through a reduction in BCAA catabolism. Additionally, this combined supplementation strategy demonstrated potential benefits in improving aerobic endurance and may contribute to the attenuation of exercise-induced central fatigue. Full article

(This article belongs to the Special Issue Interactions of Diet, Exercise, and Metabolism)

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15 pages, 873 KB

Open AccessArticle

Neck Circumference as a Practical Anthropometric Biomarker for Visceral Adiposity and Metabolic Dysregulation in Type 2 Diabetes

by Meixia Ji, Zhifu Zeng, Zhengliang Huang, Zhaowei Shi and Meifen Ji

Metabolites 2026, 16(2), 93; https://doi.org/10.3390/metabo16020093 - 26 Jan 2026

Abstract

Objective: Visceral adipose tissue is a primary driver of insulin resistance and dysglycemia in type 2 diabetes (T2D), yet its clinical assessment remains challenging. This study aimed to validate neck circumference (NC) as a novel, practical anthropometric biomarker for estimating visceral fat area [...] Read more.

Objective: Visceral adipose tissue is a primary driver of insulin resistance and dysglycemia in type 2 diabetes (T2D), yet its clinical assessment remains challenging. This study aimed to validate neck circumference (NC) as a novel, practical anthropometric biomarker for estimating visceral fat area (VFA) and identifying metabolic risk in a T2D cohort, facilitating its integration into public health and primary care screening strategies. Methods: In a cross-sectional study of 1139 T2D patients, we collected data on NC, biochemical parameters (fasting plasma glucose, hemoglobin A1c, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides), and precisely measured VFA and subcutaneous fat area (SFA) via bioelectrical impedance analysis (Omron HDS-2000). We employed Pearson’s correlation and multivariate logistic regression to analyze the relationship between NC and metabolic indicators. Receiver operating characteristic (ROC) curve analysis was used to establish sex-specific NC cut-off values for predicting abnormal VFA. Results: The cohort comprised 687 (60.3%) males and 452 (39.7%) females. NC demonstrated strong positive correlations with VFA (p < 0.001), as did body mass index (BMI), waist–hip ratio (WHR), and SFA. In males, NC was further positively correlated with key metabolic biomarkers including fasting insulin, Insulin Resistance Index, triglycerides, and creatinine. ROC analysis identified NC > 39.5 cm for males and >35.5 cm for females as the optimal cut-off points for detecting abnormal visceral adiposity, highlighting its diagnostic utility. Conclusions: NC serves as a highly accessible and effective biomarker for visceral adiposity and associated metabolic dysfunction in patients with T2D. The established sex-specific cut-off values provide a simple, non-invasive tool for risk stratification in clinical and public health settings, enabling early intervention and improved management of metabolic disease. Full article

(This article belongs to the Special Issue Advancing Metabolic/Microbial Biomarker Applications in Disease Prevention, Diagnosis and Public Health)

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27 pages, 2469 KB

Open AccessReview

The “Immune Rebellion” from the Intestines to the Liver: A Vicious Cycle That Causes the Liver to Collapse

by Wan-Ting Wang, Jia-Le Tian, Shuo Gao, Mao-Bing Wang, Yang Luo and Xun Li

Metabolites 2026, 16(2), 92; https://doi.org/10.3390/metabo16020092 - 25 Jan 2026

Abstract

The gut immune microenvironment and the liver engage in intricate information exchange via the gut–liver axis. The disruption of these interactions plays a pivotal role in the formation and exacerbation of pathological damage to the liver. The gut immune microenvironment is not an [...] Read more.

The gut immune microenvironment and the liver engage in intricate information exchange via the gut–liver axis. The disruption of these interactions plays a pivotal role in the formation and exacerbation of pathological damage to the liver. The gut immune microenvironment is not an independent layer of the gut barrier; rather, it permeates and regulates all other barrier functions, serving as the core coordinator. Disruption of the immune microenvironment in the gut–liver axis drives progression across the full disease spectrum—from steatosis to hepatitis, fibrosis, and even liver cancer—through the continuous influx of immune-stimulatory signals that overwhelm the liver’s intrinsic immune regulatory mechanisms. Dysfunction of innate immunity components, amplification of inflammatory factors and key cellular signaling pathways, activation of adaptive immune T cells, and systemic effects mediated by liver-derived inflammatory factors collectively form a disordered immune microenvironment. This damages the intestinal barrier and exacerbates liver disease via the gut–liver axis, leading to further intestinal injury, thus establishing a self-reinforcing vicious cycle. Current therapeutic strategies based on modulating the gut–liver axis microenvironment remain limited, yet studies have demonstrated that suppressing gut immune cells, cytokines, and signaling pathways can help delay liver disease progression. Hopefully, future combined, precise, and cutting-edge gut immunotherapies will provide more effective strategies for liver disease treatment. Full article

(This article belongs to the Section Thematic Reviews)

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15 pages, 267 KB

Open AccessArticle

Body Adiposity Indices, Adipokines Profile, and CNR1 Polymorphisms in Atypical Phenotypes of Obesity

by Simona Georgiana Popa, Loredana Maria Marin, Loredana Maria Dira, Ana Cristina Tudosie and Andreea Loredana Golli

Metabolites 2026, 16(2), 91; https://doi.org/10.3390/metabo16020091 - 25 Jan 2026

Abstract

Background/Objectives: Insulin-Resistant Normal Weight and Insulin-Sensitive Obesity are atypical cardiometabolic phenotypes whose clinico-biological features, management, and prognosis are a subject of extensive scientific debate. The current study aimed to assess the prevalence of metabolic phenotypes of obesity and to evaluate their association with [...] Read more.

Background/Objectives: Insulin-Resistant Normal Weight and Insulin-Sensitive Obesity are atypical cardiometabolic phenotypes whose clinico-biological features, management, and prognosis are a subject of extensive scientific debate. The current study aimed to assess the prevalence of metabolic phenotypes of obesity and to evaluate their association with markers related to diabesity, adipokines profile, and two single nucleotide polymorphisms of CNR1 gene. Methods: We performed a cross-sectional analysis in a random sample of 487 individuals (53.03 ± 13.71 years, 48.3% male) which were classified based on body mass index (</≥25 kg/m²) and insulin resistance (HOMA-IR cut-off value 2.5) as Insulin-Sensitive/Insulin-Resistant Normal Weight (ISNW/IRNW) and Insulin-Sensitive/Insulin-Resistant Obesity (ISO/IRO). Results: The ISO phenotype frequency was 24.2%, with a higher prevalence in the 40–60 years age group (47.0%) and in men (44.9%), while the prevalence of IRNW was 7.0%, predominating in women (61.8%). Participants with IRNW had a more altered glycoregulation profile (fasting and 2 h OGTT blood glucose, prediabetes, and hyperinsulinism), hypercholesterolemia, and adiposity indices (ABSI) than those with ISNW, but comparable to those with IRO. Participants with ISO had a more favorable glycoregulation profile, lipid profile, adipocytokines, and adiposity indices than those with IRO. IRNW had higher odds of being associated with prediabetes (OR 10.75; p < 0.001) than ISNW, while younger age, CUN-BAE, and ABSI were independently associated with both ISO and IRNW phenotypes. Conclusions: The IRNW phenotype should be actively evaluated to intervene on the cardiometabolic risk, while further studies are needed to confirm the sustainability of the favorable cardiometabolic profile of the ISO phenotype. Full article

(This article belongs to the Special Issue Metabolic Signature of Type 2 Diabetes, Insulin Resistance, and Obesity Triad)

13 pages, 628 KB

Open AccessReview

Metabolic and Anthropometric Alterations in Juvenile Idiopathic Arthritis: A Focus on Cardiometabolic Risk and Non-Invasive Evaluation Methods

by Agnieszka Januś, Justyna Roszkiewicz and Elżbieta Smolewska

Metabolites 2026, 16(2), 90; https://doi.org/10.3390/metabo16020090 - 24 Jan 2026

Abstract

Juvenile idiopathic arthritis (JIA) is the most prevalent chronic rheumatologic condition in childhood, with an incidence that continues to rise worldwide. Despite substantial progress in therapeutic strategies over the past two decades, JIA remains a major health concern. Beyond joint inflammation and musculoskeletal [...] Read more.

Juvenile idiopathic arthritis (JIA) is the most prevalent chronic rheumatologic condition in childhood, with an incidence that continues to rise worldwide. Despite substantial progress in therapeutic strategies over the past two decades, JIA remains a major health concern. Beyond joint inflammation and musculoskeletal impairment, accumulating evidence indicates that JIA is associated with metabolic disturbances and altered body composition, which may predispose affected children to an elevated cardiovascular risk in the long term. The objective of this review is to synthesize current knowledge on these metabolic and anthropometric alterations and to evaluate the role of non-invasive diagnostic methods in detecting early cardiovascular changes. A narrative review of the literature was conducted using PubMed and Scopus databases, focusing on studies assessing lipid metabolism, insulin resistance, adiposity, and cardiovascular markers in pediatric patients with JIA. Special attention was given to non-invasive diagnostic approaches, including bioelectrical impedance analysis (BIA), dual-energy X-ray absorptiometry (DXA), skinfold thickness, transient elastography, carotid intima–media thickness (cIMT), as well as selected biochemical markers. Evidence suggests that children with JIA frequently present with dyslipidemia, increased insulin resistance, and abnormal body fat distribution compared with their healthy peers. Non-invasive assessment methods, particularly DXA and cIMT, have demonstrated sensitivity in detecting subclinical metabolic and vascular changes. These alterations resemble early features of metabolic syndrome and are thought to contribute to premature cardiovascular morbidity in this population. Incorporating non-invasive cardiovascular risk assessment into routine rheumatology practice may improve early detection of metabolic and vascular complications in JIA, support timely preventive interventions, and ultimately enhance long-term outcomes for affected children. Most available evidence is derived from cross-sectional studies, highlighting the need for longitudinal investigations to better define long-term cardiometabolic risk in JIA. Full article

(This article belongs to the Special Issue The Metabolic Genesis of Cardiovascular Disease)

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1 pages, 131 KB

Open AccessCorrection

Correction: Song et al. Serum Uric Acid and Bone Health in Middle-Aged and Elderly Hypertensive Patients: A Potential U-Shaped Association and Implications for Future Fracture Risk. Metabolites 2025, 15, 15

by Shuaiwei Song, Xintian Cai, Junli Hu, Qing Zhu, Di Shen, Huimin Ma, Yingying Zhang, Rui Ma, Pan Zhou, Wenbo Yang, Jing Hong and Nanfang Li

Metabolites 2026, 16(1), 89; https://doi.org/10.3390/metabo16010089 - 22 Jan 2026

Abstract

References [...] Full article

(This article belongs to the Special Issue Nutrition and Metabolic Changes in Aging and Age-Related Diseases)

18 pages, 1890 KB

Open AccessSystematic Review

The Effects of High-Intensity Interval Training on Inflammatory Cytokines in Children and Adolescents with Obesity: A Systematic Review and Meta-Analysis

by Meng Cao, Pei Sun, Xiaodong Wang and Mengxian Zhao

Metabolites 2026, 16(1), 88; https://doi.org/10.3390/metabo16010088 - 21 Jan 2026

Abstract

Background: High-intensity interval training (HIT) is a time-efficient strategy to improve metabolic health in children, but its impact on inflammatory markers is still unclear. Therefore, we conducted a meta-analysis to examine the role of HIT on pro-inflammatory cytokines including C-reactive protein (CRP), [...] Read more.

Background: High-intensity interval training (HIT) is a time-efficient strategy to improve metabolic health in children, but its impact on inflammatory markers is still unclear. Therefore, we conducted a meta-analysis to examine the role of HIT on pro-inflammatory cytokines including C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) in children with overweight/obesity. Methods: A meta-analysis was conducted following PRISMA guidelines. PubMed, Web of Science, Scopus, and Embase were searched up to 31 July 2025, for studies involving children with overweight/obesity aged 6 to 18 years. Randomized controlled trials and non-randomized controlled trials with outcome measurements that included CRP, IL-6, and TNF-α were included. Random-effects models were used to aggregate a mean effect size (ES) with 95% confidence intervals (CI), and potential moderators were explored. Results: In total, 768 participants from 15 studies were included. HIT significantly improved CRP (574 participants, 13 studies, SMD = −0.63, 95% CI: −1.02 to −0.24, p < 0.01) when compared to control group/pre-intervention. There were no significant effects on IL-6 and TNF-α, and no differences when compared to moderate-intensity training. Subgroup analyses indicated greater effectiveness in intervention duration, work-and-rest ratio, and work time were the significant moderators (p < 0.05). Conclusions: High-intensity interval training is effective for reducing CRP levels in children with obesity. Intervention duration, work-and-rest ratio, and work time can affect the intervention effects of HIT. Full article

(This article belongs to the Special Issue Adipose Thermogenesis and Crosstalk: Signaling Networks in Obesity, Metabolic Disease and Beyond)

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23 pages, 797 KB

Open AccessReview

Metabolic Bone Disease in Captive Flying Foxes: A Conceptual Framework and Future Perspectives

by Diana Faim, Isabel Pires and Filipe Silva

Metabolites 2026, 16(1), 87; https://doi.org/10.3390/metabo16010087 - 21 Jan 2026

Abstract

In Pteropus spp., metabolic bone disease has been consistently associated with fruit-based diets that are deficient in calcium, vitamin D precursors, and protein, as well as limited ultraviolet B (UVB) exposure, as reported in zoological surveys and clinical observations. Comparative mammalian physiology suggests [...] Read more.

In Pteropus spp., metabolic bone disease has been consistently associated with fruit-based diets that are deficient in calcium, vitamin D precursors, and protein, as well as limited ultraviolet B (UVB) exposure, as reported in zoological surveys and clinical observations. Comparative mammalian physiology suggests that dysregulation of the endocrine axis involving parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), calcitonin, and calcitriol may contribute to disease development, although direct species-specific endocrine data in flying foxes remain scarce. This narrative review synthesizes current knowledge from published zoological reports, clinical observations, and comparative mammalian physiology regarding the etiology, pathophysiology, and clinical expression of metabolic bone disease in captive flying foxes. Much of the available evidence is derived from juvenile Pteropus vampyrus, and its applicability to other Pteropus species remains to be fully established. The limited availability and consistency of existing data, together with the scarcity of controlled experimental and longitudinal studies, necessarily constrain the conclusions that can be drawn. Nevertheless, this review highlights key nutritional and environmental risk factors and summarizes evidence-informed preventive management strategies to improve skeletal health and welfare in managed Pteropus populations. Full article

(This article belongs to the Special Issue Nutritional and Metabolic Influences on Animal Growth and Reproduction)

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20 pages, 1702 KB

Open AccessArticle

Artificial Neural Network Elucidates the Role of Transport Proteins in Rhodopseudomonas palustris CGA009 During Lignin Breakdown Product Catabolism

by Niaz Bahar Chowdhury, Mark Kathol, Nabia Shahreen and Rajib Saha

Metabolites 2026, 16(1), 86; https://doi.org/10.3390/metabo16010086 - 21 Jan 2026

Abstract

Background: Rhodopseudomonas palustris is a metabolically versatile bacterium with significant biotechnological potential, including the ability to catabolize lignin and its heterogeneous breakdown products. Understanding the molecular determinants of growth on lignin-derived compounds is essential for advancing lignin valorization strategies under both aerobic [...] Read more.

Background: Rhodopseudomonas palustris is a metabolically versatile bacterium with significant biotechnological potential, including the ability to catabolize lignin and its heterogeneous breakdown products. Understanding the molecular determinants of growth on lignin-derived compounds is essential for advancing lignin valorization strategies under both aerobic and anaerobic conditions. Methods: R. palustris was cultivated on multiple lignin breakdown products (LBPs), including p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, p-coumarate, sodium ferulate, and kraft lignin. Condition-specific transcriptomics and proteomics datasets were generated and used as input features to train machine-learning models, with experimentally measured growth rates as the prediction target. Artificial Neural Networks (ANNs), Random Forest (RF), and Support Vector Machine (SVM) models were evaluated and compared. Permutation feature importance analysis was applied to identify genes and proteins most influential for growth. Results: Among the tested models, ANNs achieved the highest predictive performance, with accuracies of 94% for transcriptomics-based models and 96% for proteomics-based models. Feature importance analysis identified the top twenty growth-associated genes and proteins for each omics layer. Integrating transcriptomic and proteomic results revealed eight key transport proteins that consistently influenced growth across LBP conditions. Re-training ANN models using only these eight transport proteins maintained high predictive accuracy, achieving 86% for proteomics and 76% for transcriptomics. Conclusions: This study demonstrates the effectiveness of ANN-based models for predicting growth-associated genes and proteins in R. palustris. The identification of a small set of key transport proteins provides mechanistic insight into lignin catabolism and highlights promising targets for metabolic engineering aimed at improving lignin utilization. Full article

(This article belongs to the Section Cell Metabolism)

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14 pages, 2631 KB

Open AccessArticle

Comparative Analysis of Chemical Constituents in Peppers from Different Regions by Integrated LC-MS and GC-MS Non-Targeted Metabolomics

by Xuefeng Gong, Sihao Hou, Yi Xu, Hong Li, Xin Chen and Zhanfeng Song

Metabolites 2026, 16(1), 85; https://doi.org/10.3390/metabo16010085 - 21 Jan 2026

Abstract

Background/Objectives: The quality of dried chili peppers is critically influenced by geographical origin, yet the metabolic basis for these differences remains insufficiently explored. This study sought to elucidate the region-specific metabolic profiles and their association with key quality traits in the pepper cultivar [...] Read more.

Background/Objectives: The quality of dried chili peppers is critically influenced by geographical origin, yet the metabolic basis for these differences remains insufficiently explored. This study sought to elucidate the region-specific metabolic profiles and their association with key quality traits in the pepper cultivar ‘Hong Guan 6’. Methods: Fruits harvested from three major cultivation regions in China were analyzed. We quantified fat and capsaicinoid content and employed an integrated LC-MS and GC-MS untargeted metabolomics approach to characterize the metabolite composition. Multivariate statistical analyses were applied to identify differentially abundant metabolites (DAMs) and uncover their related biochemical pathways. Results: Significant regional variations in fat and capsaicinoid content were observed, with peppers from Pengzhou (PZ) exhibiting the highest capsaicin levels. Metabolomic profiling revealed 529 metabolites that were significantly more abundant in PZ samples. These metabolites were enriched in several key pathways, including beta-alanine metabolism, plant hormone signal transduction, and N-glycan biosynthesis. Specifically, elevated levels of β-alanine and malonate in the beta-alanine metabolism pathway were detected in PZ and Anyue (AY) samples, suggesting a potential biochemical mechanism for their enhanced fat synthesis. Conclusions: Our findings demonstrate that geographical origin significantly reprograms the pepper metabolome, directly impacting quality attributes. The results provide crucial insights into the biochemical mechanisms, particularly those involving beta-alanine metabolism, that underpin the differences in critical quality traits such as fat content. Full article

(This article belongs to the Special Issue Advances in Food Sciences: Metabolomics to Unravel the Complexity of Food Metabolites)

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21 pages, 2287 KB

Open AccessArticle

Chemical Attributes of UK-Grown Tea and Identifying Catechin and Metabolite Dynamics in Green and Black Tea Using Metabolomics and Machine Learning

by Amanda J. Lloyd, Jasen Finch, Alina Warren-Walker, Alison Watson, Laura Lyons, MJ Pilar Martinez Martin, Thomas Wilson and Manfred Beckmann

Metabolites 2026, 16(1), 84; https://doi.org/10.3390/metabo16010084 - 21 Jan 2026

Abstract

The Dartmoor Estate Tea plantation in Devon, UK, benefits from a unique microclimate and diverse soil conditions, which, together with its different processing methods, contribute to the distinctive flavours and chemical profiles of its teas. Objectives: The chemical diversity of Dartmoor tea was [...] Read more.

The Dartmoor Estate Tea plantation in Devon, UK, benefits from a unique microclimate and diverse soil conditions, which, together with its different processing methods, contribute to the distinctive flavours and chemical profiles of its teas. Objectives: The chemical diversity of Dartmoor tea was assessed via samples collected during processing of green and black tea. Methods: Leaf samples were collected during the processing of green and black tea and analysed using Flow Infusion Electrospray Ionisation Mass Spectrometry (FIE-MS). Results: For green tea processing, random forest regression identified features associated with the processing steps, resulting in a total of 272 m/z explanatory features. The analysis of black tea processing (4 h and overnight oxidation prior to roasting) yielded 209 discriminatory m/z features (4 h) and the model for the overnight oxidation and roasting treatments yielded 605 discriminatory m/z features. K-means clustering was performed on the percentage of relative abundance of the discriminatory m/z features. This grouped the discriminatory m/z features into 15 clusters of features showing similar trends across the processing stages. Functional and structural enrichment analysis was performed on each of the clusters and significant metabolic pathways included metabolism and biosynthesis of flavonoids, amino acids and lipids, the Pentose phosphate pathway, and the TCA cycle. Many discriminatory features were putatively classified as catechin-derived flavan-3-ols and flavonol glycosides. Conclusions: This research highlights the complex role that processing plays in shaping tea quality. It provides valuable insights into the metabolic pathways that influence tea production and emphasises how these factors determine the final chemical profile and sensory characteristics of tea. Full article

(This article belongs to the Section Food Metabolomics)

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14 pages, 497 KB

Open AccessArticle

Vitamin D Status in People Living with HIV: Assessment of 25(OH)D Levels and Associated Factors—A Cross-Sectional Study

by Florentina Dumitrescu, Eugenia-Andreea Marcu, Vlad Pădureanu, Livia Dragonu, Ilona-Andreea Georgescu, Lucian Giubelan, Rodica Pădureanu and Sineta Cristina Firulescu

Metabolites 2026, 16(1), 83; https://doi.org/10.3390/metabo16010083 - 21 Jan 2026

Abstract

Background: Vitamin D deficiency (VDD) is highly prevalent among people living with human immunodeficiency virus (HIV), with reported rates of insufficiency and deficiency substantially higher than in many general-population cohorts. This study aims to assess the prevalence of vitamin D deficiency and to [...] Read more.

Background: Vitamin D deficiency (VDD) is highly prevalent among people living with human immunodeficiency virus (HIV), with reported rates of insufficiency and deficiency substantially higher than in many general-population cohorts. This study aims to assess the prevalence of vitamin D deficiency and to investigate the risk factors contributing to its occurrence among people living with HIV who are receiving antiretroviral therapy (ART) and are registered at the Craiova Regional Center (CRC). Methods: A retrospective study was conducted from May 2024 to August 2024, including individuals with HIV aged 18 years and older who were registered at the CRC. Results: A total of 138 patients were included in the study. The prevalence of vitamin D deficiency (<20 ng/mL) and vitamin D insufficiency (20–29.9 ng/mL) was 36.2% and 33.3%, respectively, with an average vitamin D level of 26.4 ± 9.9 ng/mL. Vitamin D deficiency was associated with obesity (p = 0.0013), high HIV viral load (p = 0.043), low CD4 nadir (<200 cells/mm³, p = 0.006), prolonged ART exposure (p = 0.002), and the use of tenofovir disoproxil fumarate or protease inhibitor-containing regimens (p = 0.034 and p = 0.016, respectively). Conclusions: These findings indicate that monitoring vitamin D levels could be particularly relevant for patients with HIV with higher-risk profiles. However, our study included a relatively small number of participants, so further research in larger cohorts is needed to better understand these patterns. Full article

(This article belongs to the Special Issue Vitamin D Metabolism and Human Health)

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14 pages, 3496 KB

Open AccessArticle

Distinct Metabolic Signatures Linked to High-Resolution Computed Tomography Radiographic Phenotypes in Stable and Progressive Fibrotic Lung Disease

by Girish B. Nair, Faizan Faizee, Zachary Smith, Sayf Al-Katib, Nadia Ashrafi, Ali Yilmaz, Romana Ashrafi Mimi, Sarayu Bhogoju, Vilija Lomeikaite, Juozas Gordevičius, Edward Castillo and Stewart F. Graham

Metabolites 2026, 16(1), 82; https://doi.org/10.3390/metabo16010082 - 19 Jan 2026

Abstract

Background: This study aimed to identify distinct metabolic signatures associated with disease progression by integrating high-resolution computed tomography (HRCT) visual scoring with comprehensive metabolomic profiling. Materials and Methods: This single-center, cross-sectional study enrolled 60 idiopathic pulmonary fibrosis/interstitial lung disease (IPF/ILD) patients [...] Read more.

Background: This study aimed to identify distinct metabolic signatures associated with disease progression by integrating high-resolution computed tomography (HRCT) visual scoring with comprehensive metabolomic profiling. Materials and Methods: This single-center, cross-sectional study enrolled 60 idiopathic pulmonary fibrosis/interstitial lung disease (IPF/ILD) patients with usual interstitial pneumonia pattern. Participants underwent standardized pulmonary function testing, HRCT imaging, and peripheral blood collection for metabolomic analysis using one-dimensional hydrogen nuclear magnetic resonance spectroscopy and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. Linear regression analysis integrated radiographic scores with metabolomic profiles, adjusted for multiple covariates. Results: Stable IPF/ILD exhibited moderate negative correlations between the six most significant metabolites and HRCT scores (r = −0.27 to −0.51), along with a high abundance of specific phospholipids (triacylglycerol, monoacylglycerol, phosphatidylglycerol, phosphatidylethanolamine, diacylglycerol), sphingomyelin, ceramide, and acylcarnitine. In contrast, progressive disease showed weak positive correlations between the six most significant metabolites and HRCT scores (r = 0.19–0.26), and moderate negative correlation between specific triacylglycerol species and HRCT scores (r = −0.37–0.4). Furthermore, metabolomic analysis in individuals with progressive disease revealed both high and low abundances of specific phospholipid species (including high and low triacylglycerol species, as well as low levels of phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol), along with high levels of certain sphingomyelin, ceramide, taurine, and purine bases, and low levels of xanthine and lactic acid observed. Conclusions: Integration of systematic HRCT semi-quantitative scoring with metabolomic profiling successfully differentiated stable from progressive IPF/ILD through distinct molecular-radiographic signatures. Full article

(This article belongs to the Special Issue State-of-the-Art NMR-Based Metabolomics and Its Applications)

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31 pages, 1713 KB

Open AccessArticle

In Vitro Antioxidant, Anti-Platelet and Anti-Inflammatory Natural Extracts of Amphiphilic Bioactives from Organic Watermelon Juice and Its By-Products

by Emmanuel Nikolakakis, Anna Ofrydopoulou, Katie Shiels, Sushanta Kumar Saha and Alexandros Tsoupras

Metabolites 2026, 16(1), 81; https://doi.org/10.3390/metabo16010081 - 19 Jan 2026

Abstract

Background/Objectives: Watermelon (Citrullus lanatus) processing generates substantial quantities of rind, seeds, and residual pulp that are typically discarded despite being rich in polyunsaturated fatty acids, polar lipids, carotenoids, and phenolic compounds. These amphiphilic bioactives are increasingly recognized for their roles in [...] Read more.

Background/Objectives: Watermelon (Citrullus lanatus) processing generates substantial quantities of rind, seeds, and residual pulp that are typically discarded despite being rich in polyunsaturated fatty acids, polar lipids, carotenoids, and phenolic compounds. These amphiphilic bioactives are increasingly recognized for their roles in modulating oxidative stress, inflammation, and platelet activation; however, the lipid fraction of watermelon by-products remains insufficiently characterized. This study examined organic watermelon juice and its by-products to isolate, characterize, and evaluate extracts enriched in amphiphilic and lipophilic bioactives, with emphasis on their in vitro antioxidant, anti-inflammatory, and antithrombotic properties. Methods: total lipids were extracted using a modified Bligh–Dyer method and fractionated into total amphiphilic compounds (TAC) and total lipophilic compounds (TLC) via counter-current distribution. Phenolic and carotenoid levels were quantified, and antioxidant capacity was assessed using DPPH, ABTS, and FRAP assays. Anti-platelet and anti-inflammatory activities were evaluated against ADP- and PAF-induced platelet aggregation. Structural characterization of polar lipids was performed using ATR–FTIR, and LC–MS was used to determine fatty acid composition and phospholipid structures. Results and Discussion: Carotenoids were primarily concentrated in the TLC fractions with high ABTS values for antioxidant activity, while phenolics mostly in the juice, the TACs of which showed the strongest total antioxidant capacity based on DPPH. TAC fractions of both samples showed also higher FRAP values of antioxidant activity, likely due to greater phenolic content. TAC extracts also exhibited notable inhibition of PAF- and ADP-induced platelet aggregation, associated with their enriched ω-3 PUFA profiles and favorable ω-6/ω-3 ratios based on their LC-MS profiles. Conclusions: Overall, watermelon products (juice) and by-products represent a valuable and sustainable source of amphiphilic bioactives with significant antioxidant, anti-inflammatory, and anti-platelet potential, supporting their future use in functional foods, nutraceuticals, and cosmetic applications. Full article

(This article belongs to the Special Issue Secondary Metabolites and Their Activities: From the Identification to the Biological Investigation)

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