Sodium-glucose co-transporter 2 inhibitors (SGLT2i), when combined with metformin (COMBI), offer multi-organ protective effects in patients with type 2 diabetes (T2D), particularly those at high risk of cardiovascular or renal complications. However, the underlying molecular mechanisms remain poorly understood. We profiled 303 targeted serum metabolites in 1494 participants of the KORA study, including T2D patients treated with COMBI therapy, metformin monotherapy, or no glucose-lowering medication. Additionally, metabolomic profiling was quantified on seven tissues (plasma, liver, adrenal glands, adipose tissue, testis, lung, and cerebellum), and related hepatic transcripts were evaluated in 40 mice. Multivariable linear regression analyses, adjusted for age, sex, BMI, lifestyle, glycemic, and cardiovascular risk factors, were applied to human data; tissue-specific regression analyses were conducted for murine samples. Identified metabolites were further investigated using biochemical pathway analyses and literature review. COMBI therapy was associated with significant changes in metabolite profiles. In humans, 10 metabolites were significantly altered compared to metformin monotherapy. In mice, 82 altered metabolites were identified in plasma, 52 in liver, 30 in adrenal glands, 12 in adipose tissue, seven in testis, seven in lung, and six in cerebellum. COMBI therapy lowered threonine concentrations in both human serum and murine plasma but raised threonine, glycine, and urea cycle metabolites (citrulline, asymmetric dimethyl arginine (ADMA), and ornithine) in murine liver. This was accompanied by enhanced hepatic expression of Slc38a2, a threonine transporter gene. In humans, urea cycle metabolites correlated strongly with the fibrosis-4 index, a marker of liver fibrosis. Additionally, COMBI therapy elevated ketone body markers, such as hydroxybutyrylcarnitine, across murine liver, plasma, adrenal glands, adipose tissue, and testis. COMBI therapy modulates amino acid metabolism, the urea cycle, and ketone body production, suggesting potential mechanisms underlying its protective effects against liver fibrosis and male subfertility. These findings provide novel insights into the systemic metabolic actions of COMBI therapy and highlight its translational potential to improve clinical outcomes in T2D patients.

RNA binding proteins have multiple diverse cellular functions and are often mis-regulated in disease. Despite their many cellular functions and implications in disease, very little is known about their physiological functions. Here we describe a novel zebrafish knockout model of the RNA binding proteins Hnrnpa1 and Hnrnpa3. Loss of Hnrnpa3 in zebrafish has no obvious morphological phenotype. Similarly, single mutants of the duplicated zebrafish hnrnpa1 genes, hnrnpa1a and hnrnpa1b, have no discernible phenotype, whereas the hnrnpa1a; hnrnpa1b double mutants are embryonic lethal. They display muscle, vascular and developmental defects with a reduced volume of the yolk extension. Metabolic profiling revealed severe changes in lipid metabolism in the hnrnpa1a; hnrnpa1b double mutants. Our analysis identified the involvement of Hnrnpa1 in many cellular pathways including the regulation of lipid metabolism and opens the door for future therapeutic studies in HNRNPA-associated diseases.

OBJECTIVE:Villus growth in the small bowel by Glucagon-like peptide-2 (GLP-2) pharmacotherapy improves intestinal absorption capacity and is now used clinically for the treatment of short bowel syndrome and intestinal failure occurring after extensive intestinal resection. Another recently acknowledged effect of GLP-2 treatment is the inhibition of gallbladder motility and increased gallbladder refilling. However, the impact of these two GLP-2-characteristic effects on bile acid metabolism in health and after intestinal resection is not understood. METHODS:Mice were injected with the GLP-2-analogue teduglutide or vehicle. We combined the selenium-75-homocholic acid taurine (SeHCAT) assay with novel spatial imaging in healthy mice and after ileocecal resection (ICR mice) and associated the results with clinical stage targeted bile acid metabolomics as well as gene expression analyses. RESULTS:ICR mice had virtual complete intestinal loss of secondary bile acids, and an increased ratio of 12α-hydroxylated vs. non-12α-hydroxylated bile acids, which was attenuated by teduglutide. Teduglutide promoted SeHCAT retention in healthy and in ICR mice. Acute concentration of the SeHCAT-signal into the hepatobiliary system was observed. Teduglutide induced significant repression of hepatic cyp8b1 expression, likely by induction of MAF BZIP Transcription Factor G. CONCLUSIONS:The data suggest that GLP-2-pharmacotherapy in mice significantly slows bile acid circulation primarily via hepatic Farnesoid X receptor-signaling.

Purpose To compare the plasma metabolic profile of patients with a CRB1-associated inherited retinal degeneration (CRB1-IRD) with healthy controls (HCs). Design A case-control study. Participants A cohort of 30 Dutch CRB1-associated IRD patients and 29 Dutch healthy controls. Methods The MxP® Quant 500 Kit was used for measuring metabolite concentrations. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in μM (μmol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls. Main Outcome Measures Plasma concentration of 619 metabolites. Results Over-representation of pathways among metabolites associated strongest to CRB1-IRDs (P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes. Conclusions Lipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1-IRD.

BACKGROUND:Childhood asthma has been linked to distinct metabolomic profiles. OBJECTIVE:We sought to identify phenotypes (metabotypes) in children with moderate to severe asthma through integrative fecal and serum metabolome analysis. METHODS:Children from the Systems Pharmacology Approach to Uncontrolled Pediatric Asthma cohort with Global Initiative for Asthma treatment step 3 or higher were recruited. Asthma control was defined by the Asthma Control Test and annual exacerbation history. Targeted metabolomic profiling of feces and serum was performed using liquid chromatography and flow injection electrospray ionization-triple quadrupole mass spectrometry. Similarity network fusion integrated fecal and serum metabolome profiles, followed by spectral clustering. Clusters were analyzed for differences in asthma characteristics, food diaries, fecal microbiota composition, and levels of serum inflammatory markers and blood cells. RESULTS:Integrative fecal and serum metabolome analysis of 92 children with moderate to severe asthma (median age, 11.5 years, 34% female) revealed 3 metabotypes. Metabotype 1 had the lowest percentage of allergic rhinitis, with elevated serum ceramides and triglycerides. Metabotype 2 had higher odds of asthma control, the highest percentage of children with 4 or more months of breast-feeding, reduced sugar intake, lowest levels of blood neutrophils and serum inflammatory markers, and elevated serum acylcarnitines and ω-3 fatty acids. Metabotype 3 included the highest percentage of uncontrolled asthma patients, with decreased serum cholesteryl esters, phosphatidylcholines, and sphingomyelins, elevated fecal amino acids, and reduced fecal microbiota diversity. CONCLUSIONS:Metabotypes in children with moderate to severe asthma are linked to asthma control, distinct fecal microbiota, and systemic inflammatory patterns. The findings suggest that metabotyping can be valuable in precision medicine approaches for asthma.

INTRODUCTION:Since the early 2000s, metabolomics has grown rapidly, becoming integral to fields like life sciences, health, and environmental research. This expansion has led to the formation of national and international societies, such as Germany's DGMet, to tackle emerging challenges. One of DGMet's goals is to improve measurement quality by assessing community needs for harmonization and standardization. A recent survey within the German-speaking community aimed to identify current practices and gaps in the use of chemical standards and reference materials, to guide future standardization efforts and collaborative initiatives. METHODS:An online survey was conducted between June 2023 and April 2024. The survey consisted of 38 key questions and was open to research institutions from Germany, Austria, and Switzerland. RESULTS:The survey was accessed by 68 laboratories, with 23 institutes providing complete or partial responses (34% response rate), which is comparable to rates reported in similar surveys within the metabolomics and lipidomics communities. Respondents were mainly experienced researchers from Germany, focusing mainly on health-related ("red") metabolomics, as indicated by 78% of the respondents, followed by microbial ("grey", 48%) and plant ("green", 39%) metabolomics (multiple answers possible). The use of targeted methods was reported more frequently (91%) than that of non-targeted methods (78%), whereas metabolite fractions studied were equally split between polar, midpolar and lipid fractions (83% each). Human (74%), mouse (61%) and Arabidopsis (30%) were the most frequently studied organisms. Most participants used synthetic chemical standards for instrument qualification (83%), calibration (78%), and metabolite identification (74%), while matrix reference materials were mainly applied for quality control (52%) and method validation (44%). There was a strong demand for more standards, especially for metabolite identification and quantification, with cost being a major barrier, particularly for isotopically labelled standards and certified reference materials. CONCLUSIONS:Valuable insights into the use of standards and reference materials within the German-speaking metabolomics community were obtained. Moving forward, the community should address critical gaps in metabolomics standardization. To achieve this, it must share its knowledge, articulate its needs clearly, and actively engage in joint efforts with national metrology institutes and international standardization initiatives.

Despite recent progress in the diagnosis and treatment of advanced cancers, the overall patient treatment outcome did not substantially improve over the last years. Therefore, developing novel therapies, which may also work synergistically in combination with the conventional therapies is crucial. One promising new therapeutic approach is bacterium-mediated cancer therapy. In the current work, we describe the influence of the gut microbiome and intranasal E. coli Nissle applications on the metabolism in cancer tissues of 4T1 syngeneic tumor bearing mice. Here we found that after gut microbiome depletion and/or E. coli Nissle treatment the ratios of ADMA/Arginine, Putrescine/Ornithine and Kynurenine/Tryptophan as well as the total concentration of Carnosine, Kynurenine and H1 (synonymus for all sugars detectable) are significantly altered in tumor tissues of as the result of treatment. In conclusion, our current data show that E. coli Nissle bacteria facilitating metabolic modulation of tumors, a finding could be important for improved cancer therapy in patients.

Purpose To compare the plasma metabolic profile of patients with a CRB1 -associated inherited retinal degeneration ( CRB1 -IRD) with healthy controls (HCs).Design A case-control study.Methods Plasma concentration of 619 metabolites was measured with the MxP® Quant 500 Kit in 30 patients with a CRB1 -IRD and 29 HCs. We fitted a linear regression model with adjustments for age and sex based on the concentration of metabolites in µM (µmol/L), or on the sums and ratios of metabolites, to determine differences between patients and controls.Results Over-representation of pathways among metabolites associated strongest to CRB1 -IRDs ( P < 0.05, n = 62) identified amino acid pathways (such as beta-alanine, histidine, and glycine/serine) and bile acid biosynthesis, driven by a decrease in deoxycholic acid derivatives produced by gut microbiota. Enrichment analysis of metabolic classes across the plasma metabolic profile further identified significant positive enrichment for lipid metabolites glycerophospholipids, cholesterol esters, and ceramides, and significant depletion for bile acid metabolites. Further investigation of the sums and ratios (i.e., metabolism indicators) ascertained a significant decrease in intestinal microbial-dependent secondary bile acid classes.Conclusions Lipid metabolic alterations and decreased microbiota-related secondary bile acid concentrations indicate significant alterations in gut metabolism in patients with a CRB1 -IRD.### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementBartimeus Foundation### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:This study has the approval of the Institutional Review Board of the University Medical Center Utrecht (UMCU)I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Metabolomics and lipidomics are pivotal in understanding phenotypic variations beyond genomics. However, quantification and comparability of mass spectrometry (MS)-derived data are challenging. Standardised assays can enhance data comparability, enabling applications in multi-center epidemiological and clinical studies. Here we evaluated the performance and reproducibility of the MxP® Quant 500 kit across 14 laboratories. The kit allows quantification of 634 different metabolites from 26 compound classes using triple quadrupole MS. Each laboratory analysed twelve samples, including human plasma and serum, lipaemic plasma, NIST SRM 1950, and mouse and rat plasma, in triplicates. 505 out of the 634 metabolites were measurable above the limit of detection in all laboratories, while eight metabolites were undetectable in our study. Out of the 505 metabolites, 412 were observed in both human and rodent samples. Overall, the kit exhibited high reproducibility with a median coefficient of variation (CV) of 14.3 %. CVs in NIST SRM 1950 reference plasma were below 25 % and 10 % for 494 and 138 metabolites, respectively. To facilitate further inspection of reproducibility for any compound, we provide detailed results from the in-depth evaluation of reproducibility across concentration ranges using Deming regression. Interlaboratory reproducibility was similar across sample types, with some species-, matrix-, and phenotype-specific differences due to variations in concentration ranges. Comparisons with previous studies on the performance of MS-based kits (including the AbsoluteIDQ p180 and the Lipidyzer) revealed good concordance of reproducibility results and measured absolute concentrations in NIST SRM 1950 for most metabolites, making the MxP® Quant 500 kit a relevant tool to apply metabolomics and lipidomics in multi-center studies.

Expression of pmCiC in human cancer tissues of different origin and at metastatic sites.

BackgroundSex hormones and sex hormone-binding globulin (SHBG) may play a role in fatty liver development. We sought to examine the association of various endogenous sex hormones, including testosterone (T), and SHBG with liver fat using complementary observational and Mendelian randomization (MR) analyses.MethodsThe observational analysis included a total of 2,239 participants (mean age 60 years; 35% postmenopausal women) from the population-based KORA study (average follow-up time: 6.5 years). We conducted linear regression analysis to investigate the sex-specific associations of sex hormones and SHBG with liver fat, estimated by fatty liver index (FLI). For MR analyses, we selected genetic variants associated with sex hormones and SHBG and extracted their associations with magnetic resonance imaging measured liver fat from the largest up to date European genome-wide associations studies.ResultsIn the observational analysis, T, dihydrotestosterone (DHT), progesterone and 17α-hydroxyprogesterone (17-OHP) were inversely associated with FLI in men, with beta estimates ranging from -4.23 to -2.30 [p-value <0.001 to 0.003]. Whereas in women, a positive association of free T with FLI (β = 4.17, 95%CI: 1.35, 6.98) was observed. SHBG was inversely associated with FLI across sexes [men: -3.45 (-5.13, -1.78); women: -9.23 (-12.19, -6.28)]. No causal association was found between genetically determined sex hormones and liver fat, but higher genetically determined SHBG was associated with lower liver fat in women (β = -0.36, 95% CI: -0.61, -0.12).ConclusionOur results provide suggestive evidence for a causal association between SHBG and liver fat in women, implicating the protective role of SHBG against liver fat accumulation.

List of metabolites measured with the AbsoluteIDQ® p180 Kit

High magnification pmCiC staining (Fig. 5C) of benign prostate epithelial cells versus cancer cells.

Intracellular Ca2+ in the presence of extracellular citrate.

Method description for metabolomics analysis from Fig. 4B.

Cancer cell proliferation in the presence of extracellular citrate.

pmCiC homology structure and blocking of pmCiC by gluconate.

Krebs cycle activity in the presence of extracellular citrate and absolute citrate levels.

Introduction The role of endogenous androgens in kidney function and disease has not been extensively explored in men and women. Research design and methods We analyzed data from the observational KORA F4 study and its follow-up examination KORA FF4 (median follow-up time 6.5 years) including 1293 men and 650 peri- and postmenopausal women, not using exogenous sex hormones. We examined the associations between endogenous androgens (testosterone [T], dihydrotestosterone [DHT], free T [fT], free DHT [fDHT], and T/DHT), with estimated glomerular filtration rate (eGFR) at baseline and follow-up, prevalent, and incident chronic kidney disease (CKD) adjusting for common CKD risk factors. Results At baseline, 73 men (5.7%) and 54 women (8.4%) had prevalent CKD. Cross-sectionally, no significant associations between androgens and kidney function were observed among men. In women, elevated T (β=-1.305, [95% CI -2.290; -0.320]) and fT (β=-1.423, [95% CI -2.449; -0.397]) were associated with lower eGFR. Prospectively, 81 men (8.8%) and 60 women (15.2%) developed incident CKD. In women, a reverse J-shaped associations was observed between DHT and incident CKD (P non-linear =0.029), while higher fDHT was associated with lower incident CKD risk (odds ratio per 1 standard deviation=0.613, [95% CI 0.369; 0.971]. Among men, T/DHT (β=-0.819, [95% CI -1.413; -0.226]) and SHBG (P non-linear =0.011) were associated with eGFR at follow-up but not with incident CKD. Some associations appeared to be modified by type 2 diabetes (T2D). Conclusion Suggestive associations are observed of androgens and SHBG with kidney impairment among men and women. However, larger well-phenotyped prospective studies are required to further elucidate the potential of androgens, SHBG, and T2D as modifiable risk factors for kidney function and CKD.