Targeted Metabolomics Assay Research Articles

Schizophrenia-Like Deficits and Impaired Glutamate/Gamma-aminobutyric acid Homeostasis in Zfp804a Conditional Knockout Mice.

Zinc finger protein 804A (ZNF804A) was the first genome-wide associated susceptibility gene for schizophrenia (SCZ) and played an essential role in the pathophysiology of SCZ by influencing neurodevelopment regulation, neurite outgrowth, synaptic plasticity, and RNA translational control; however, the exact molecular mechanism remains unclear. A nervous-system-specific Zfp804a (ZNF804A murine gene) conditional knockout (cKO) mouse model was generated using clustered regularly interspaced short palindromic repeat/Cas9 technology and the Cre/loxP method. Multiple and complex SCZ-like behaviors, such as anxiety, depression, and impaired cognition, were observed in Zfp804a cKO mice. Molecular biological methods and targeted metabolomics assay validated that Zfp804a cKO mice displayed altered SATB2 (a cortical superficial neuron marker) expression in the cortex; aberrant NeuN, cleaved caspase 3, and DLG4 (markers of mature neurons, apoptosis, and postsynapse, respectively) expressions in the hippocampus and a loss of glutamate (Glu)/γ-aminobutyric acid (GABA) homeostasis with abnormal GAD67 (Gad1) expression in the hippocampus. Clozapine partly ameliorated some SCZ-like behaviors, reversed the disequilibrium of the Glu/GABA ratio, and recovered the expression of GAD67 in cKO mice. Zfp804a cKO mice reproducing SCZ-like pathological and behavioral phenotypes were successfully developed. A novel mechanism was determined in which Zfp804a caused Glu/GABA imbalance and reduced GAD67 expression, which was partly recovered by clozapine treatment. These findings underscore the role of altered gene expression in understanding the pathogenesis of SCZ and provide a reliable SCZ model for future therapeutic interventions and biomarker discovery.

Canagliflozin regulates metabolic reprogramming in diabetic kidney disease by inducing fasting-like and aestivation-like metabolic patterns.

Sodium-glucose co-transporter 2 (SGLT2) inhibitors (SGLT2i) are antihyperglycaemic drugs that protect the kidneys of individuals with type 2 diabetes mellitus. However, the underlying mechanisms mediating the renal benefits of SGLT2i are not fully understood. Considering the fuel switches that occur during therapeutic SGLT2 inhibition, we hypothesised that SGLT2i induce fasting-like and aestivation-like metabolic patterns, both of which contribute to the regulation of metabolic reprogramming in diabetic kidney disease (DKD). Untargeted and targeted metabolomics assays were performed on plasma samples from participants with type 2 diabetes and kidney disease (n=35, 11 women) receiving canagliflozin (CANA) 100 mg/day at baseline and 12 week follow-up. Next, a systematic snapshot of the effect of CANA on key metabolites and pathways in the kidney was obtained using db/db mice. Moreover, the effects of glycine supplementation in db/db mice and human proximal tubular epithelial cells (human kidney-2 [HK-2]) cells were studied. Treatment of DKD patients with CANA for 12 weeks significantly reduced HbA1c from a median (interquartile range 25-75%) of 49.0 (44.0-57.0) mmol/mol (7.9%, [7.10-9.20%]) to 42.2 (39.7-47.7) mmol/mol (6.8%, [6.40-7.70%]), and reduced urinary albumin/creatinine ratio from 67.8 (45.9-159.0) mg/mmol to 47.0 (26.0-93.6) mg/mmol. The untargeted metabolomics assay showed downregulated glycolysis and upregulated fatty acid oxidation. The targeted metabolomics assay revealed significant upregulation of glycine. The kidneys of db/db mice undergo significant metabolic reprogramming, with changes in sugar, lipid and amino acid metabolism; CANA regulated the metabolic reprogramming in the kidneys of db/db mice. In particular, the pathways for glycine, serine and threonine metabolism, as well as the metabolite of glycine, were significantly upregulated in CANA-treated kidneys. Glycine supplementation ameliorated renal lesions in db/db mice by inhibiting food intake, improving insulin sensitivity and reducing blood glucose levels. Glycine supplementation improved apoptosis of human proximal tubule cells via the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway. In conclusion, our study shows that CANA ameliorates DKD by inducing fasting-like and aestivation-like metabolic patterns. Furthermore, DKD was ameliorated by glycine supplementation, and the beneficial effects of glycine were probably due to the activation of the AMPK/mTOR pathway.

Abstract B071: Racial differences in vaginal fluid metabolites and associations with systemic inflammation markers among ovarian cancer patients

Abstract Background: Marked racial disparity in ovarian cancer persists, even in equal-healthcare settings or after adjusting for social determinants of health, with disproportionately poor survival for Black women. This suggests an important role for biological factors. One such factor, the vaginal microbiome, is known to differ by race and to contribute to inflammation, but its potential role in ovarian cancer disparities remains poorly characterized. In this cross-sectional study, we examined whether vaginal fluid metabolites differ by race among patients with ovarian cancer, are associated with systemic inflammation, and if associations differ by race. Method: We recruited participants in a population-based setting, as part of the Ovarian Cancer Epidemiology, Healthcare Access and Disparities (ORCHiD) study, between March 2021 and September 2022. Eligible patients were of Black or White race, had a diagnosis of ovarian cancer, were aged 50 – 70 years, and provided biospecimens to ORCHiD. Overall, 36 patients were included in the study cohort, including 20 randomly selected White patients, and all 16 eligible Black patients. The main exposure variable was race, categorized into Black vs. White. We performed targeted metabolomics assays on cervicovaginal fluid. Three compound classes of metabolites were assayed for, including acylcarnitines (N=45 species), sphingomyelins (N=34), and ceramides (N=21). Five conventional metabolites were assayed for (glucose, glycerol, lactate, β-HB and total ketones). Also, we performed inflammatory assays on saliva for four cytokines (IL-1β, IL-10, TNF-α, and IL-6). Results: Seven targeted metabolites showed >2-fold change, three showed significant mean differences (at P < 0.05 but not at false discovery rate < 0.05), and two fairly distinct groups were recovered using partial least squares discriminant analysis and hierarchical clustering on metabolite concentrations. Of the 100 targeted metabolites considered, arachidonoylcarnitine (C20:4), the carnitine adduct of arachidonic acid, stood out as consistently different between Black and White. However, none of the conventional metabolites considered were significantly different by race. Approximately one-third of vaginal fluid metabolites considered and systemic inflammation biomarkers were significantly correlated, with no racial differences in correlations. Conclusion: Our findings show that vaginal fluid metabolites differed by race, with significant correlations between biomarkers of systemic inflammation and targeted metabolites. Our findings are novel and suggest that biological pathways involving mitochondrial dysfunction and sphingolipid metabolism may be relevant to ovarian cancer disparities. Future large-scale investigations are needed to verify these findings and examine their specific biological implication in the context of ovarian cancer disparity. Citation Format: Oyomoare L. Osazuwa-Peters, April Deveaux, Michael J. Muehlbauer, Olga Ilkayeva, James R. Bain, Temitope Keku, Andrew Berchuck, Bin Huang, Kevin Ward, Margaret G. Kuliszewski, Tomi Akinyemiju. Racial differences in vaginal fluid metabolites and associations with systemic inflammation markers among ovarian cancer patients [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B071.

Human Amniotic Membrane-Derived Mesenchymal Stem Cells Prevent Acute Graft-Versus-Host Disease in an Intestinal Microbiome-Dependent Manner

Acute graft-versus-host disease (aGVHD) represents a fatal severe complication after allogeneic hematopoietic stem cell transplantation. As a promising cell therapeutic strategy of aGVHD, the mechanism of mesenchymal stem cells (MSC) to ameliorate aGVHD has not been fully clarified, especially in the field of intestinal homeostasis including the intestinal microbiome involved in the pathogenesis of aGVHD. The present study aimed to explore the effect of MSC on intestinal homeostasis including the intestinal barrier and intestinal microbiome and its metabolites, as well as the role of intestinal microbiome in the preventive process of hAMSCs ameliorating aGVHD. The preventive effects of human amniotic membrane-derived MSC (hAMSCs) was assessed in humanized aGVHD mouse models. Immunohistochemistry and RT-qPCR were used to evaluate intestinal barrier function. The 16S rRNA sequencing and targeted metabolomics assay were performed to observe the alternation of intestinal microbiome and the amounts of medium-chain fatty acids (MCFAs) and short-chain fatty acids (SCFAs), respectively. Flow cytometry was performed to analyze the frequencies of T immune cells. Through animal experiments, we found that hAMSCs had the potential to prevent aGVHD. HAMSCs could repair the damage of intestinal barrier structure and function, as well as improve the dysbiosis of intestinal microbiome induced by aGVHD, and meanwhile, upregulate the concentration of metabolites SCFAs, so as to reshape intestinal homeostasis. Gut microbiota depletion and fecal microbial transplantation confirmed the involvement of intestinal microbiome in the preventive process of hAMSCs on aGVHD. Our findings showed that hAMSCs prevented aGVHD in an intestinal microbiome-dependent manner, which might shed light on a new mechanism of hAMSCs inhibiting aGVHD and promote the development of new prophylaxis regimes for aGVHD prevention.

A comprehensive investigation of the regulatory roles of OsERF096, an AP2/ERF transcription factor, in rice cold stress response.

OsERF096 negatively regulates rice cold tolerance and mediates IAA biosynthesis and signaling under cold stress. The APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors play important roles in regulating plant tolerance to abiotic stress. OsERF096 was previously identified as a direct target of miR1320, and was suggested to negatively regulate rice cold tolerance. In this study, we performed RNA-sequencing and targeted metabolomics assays to reveal the regulatory roles of OsERF096 in cold stress response. GO and KEGG analysis of differentially expressed genes showed that the starch and sucrose metabolism, plant-pathogen interaction, and plant hormone signal transduction pathways were significantly enriched. Quantification analysis confirmed a significant difference in sugar contents among WT and OsERF096 transgenic lines under cold treatment. Targeted metabolomics analysis uncovered that IAA accumulation and signaling were modified by OsERF096 in response to cold stress. Expectedly, qRT-PCR assays confirmed significant OsIAAs and OsARFs expression changes in OsERF096 transgenic lines. Finally, we identified three targets of OsERF096 based on RNA-seq, qRT-PCR, and dual-LUC assays. In summary, these results revealed the multiple regulatory roles of OsERF096 in cold stress response.

Differences in the Stool Metabolome between Vegans and Omnivores: Analyzing the NIST Stool Reference Material.

To gain confidence in results of omic-data acquisitions, methods must be benchmarked using validated quality control materials. We report data combining both untargeted and targeted metabolomics assays for the analysis of four new human fecal reference materials developed by the U.S. National Institute of Standards and Technologies (NIST) for metagenomics and metabolomics measurements. These reference grade test materials (RGTM) were established by NIST based on two different diets and two different samples treatments, as follows: firstly, homogenized fecal matter from subjects eating vegan diets, stored and submitted in either lyophilized (RGTM 10162) or aqueous form (RGTM 10171); secondly, homogenized fecal matter from subjects eating omnivore diets, stored and submitted in either lyophilized (RGTM 10172) or aqueous form (RGTM 10173). We used four untargeted metabolomics assays (lipidomics, primary metabolites, biogenic amines and polyphenols) and one targeted assay on bile acids. A total of 3563 compounds were annotated by mass spectrometry, including 353 compounds that were annotated in more than one assay. Almost half of all compounds were annotated using hydrophilic interaction chromatography/accurate mass spectrometry, followed by the lipidomics and the polyphenol assays. In total, 910 metabolites were found in at least 4-fold different levels in fecal matter from vegans versus omnivores, specifically for peptides, amino acids and lipids. In comparison, only 251 compounds showed 4-fold differences between lyophilized and aqueous fecal samples, including DG O-34:0 and methionine sulfoxide. A range of diet-specific metabolites were identified to be significantly different between vegans and omnivores, exemplified by citrinin and C17:0-acylcarnitine for omnivores, and curcumin and lenticin for vegans. Bioactive molecules like acyl alpha-hydroxy-fatty acids (AAHFA) were differentially regulated in vegan versus omnivore fecal materials, highlighting the importance of diet-specific reference materials for dietary biomarker studies.

WEAK SEED DORMANCY 1, an aminotransferase protein, regulates seed dormancy in rice through the GA and ABA pathways

Seed dormancy is a critical trait that enhances plant survival by preventing seed germination at the wrong time or under unsuitable conditions. Lack of seed dormancy in rice can lead to pre-harvest sprouting on mother plants leading to reduced yield and seed quality. Although some genes have been identified, knowledge of regulation of seed dormancy is limited. Here, we characterized a weak seed dormancy mutant named weak seed dormancy 1 (wsd1) that showed a higher seed germination percentage than the wild-type following the harvest ripeness. We cloned the WSD1 encoding an aminotransferase protein using a MutMap approach. WSD1 was stably expressed after imbibition and its protein was localized in the endoplasm reticulum. A widely targeted metabolomics assay and amino acid analysis showed that WSD1 had a role in regulating homeostasis of amino acids. PAC treatment and RNA-seq analysis showed that WSD1 regulates seed dormancy by involvement in the GA biosynthesis pathway. GA1 content and expression of GA biosynthesis-related genes were increased in the wsd1 mutant compared with the wild-type. The wsd1 mutant had reduced sensitivity to ABA. Our overall results indicated that WSD1 regulates seed dormancy by balancing the ABA and GA pathways.

Gut microbiome interacts with pregnancy hormone metabolites in gestational diabetes mellitus.

Change in the composition of intestinal microbiota is associated with metabolic disorders such as gestational diabetes mellitus (GDM). To understand how the microbiota impacts the development of gestational diabetes mellitus, we profiled the intestinal microbiome of 54 pregnant women, including 27 GDM subjects, by employing 16S rRNA gene sequencing. Additionally, we conducted targeted metabolomics assays to validate the identified pathways with overrepresented metabolites. We evaluated the patterns of changing abundances of operational taxonomic units (OTU) between GDM and the healthy counterparts over three timepoints. Based on the significant OTUs, we inferred 132 significantly altered metabolic pathways in GDM. And identified two overrepresented metabolites of pregnancy hormone, butyrate and mevalonate, as potential intermediary metabolites of intestinal microbiota in GDM. Finally, we validated the impacts of the intestinal microbiota on GDM by demonstrating consistent changes of the serum levels of progesterone, estradiol, butyrate, and mevalonate in an independent cohort. Our findings confirm that alterations in the microbiota play a role in the development of GDM by impacting the metabolism of pregnancy hormones. This provides a novel perspective on the pathogenesis of GDM and introduces potential biomarkers that can be used for early diagnosis and prevention of the disease.

Metabolite Alterations and Interactions with Microbiota in Helicobacter pylori-Associated Gastric Lesions.

Metabolites and their interactions with microbiota may be involved in Helicobacter pylori-associated gastric lesion development. This study aimed to explore metabolite alterations upon H. pylori eradication and possible roles of microbiota-metabolite interactions in progression of precancerous lesions. Targeted metabolomics assays and 16S rRNA gene sequencing were conducted to investigate metabolic and microbial alterations of paired gastric biopsy specimens in 58 subjects with successful and 57 subjects with failed anti-H. pylori treatment. Integrative analyses were performed by combining the metabolomics and microbiome profiles from the same intervention participants. A total of 81 metabolites were significantly altered after successful eradication compared to failed treatment, including acylcarnitines, ceramides, triacylglycerol, cholesterol esters, fatty acid, sphingolipids, glycerophospholipids, and glycosylceramides, with P values of <0.05 for all. The differential metabolites showed significant correlations with microbiota in baseline biopsy specimens, such as negative correlations between Helicobacter and glycerophospholipids, glycosylceramide, and triacylglycerol (P < 0.05 for all), which were altered by eradication. The characteristic negative correlations between glycosylceramides and Fusobacterium, Streptococcus, and Gemella in H. pylori-positive baseline biopsy specimens were further noticed in active gastritis and intestinal metaplasia (P < 0.05 for all). A panel including differential metabolites, genera, and their interactions may help to discriminate high-risk subjects who progressed from mild to advanced precancerous lesions in short-term and long-term follow-up periods with areas under the curve (AUC) of 0.914 and 0.801, respectively. Therefore, our findings provide new insights into the metabolites and microbiota interactions in H. pylori-associated gastric lesion progression. IMPORTANCE In this study, a panel was established including differential metabolites, genera, and their interactions, which may help to discriminate high-risk subjects for progression from mild lesions to advanced precancerous lesions in short-term and long-term follow-up.

A proposed framework to evaluate the quality and reliability of targeted metabolomics assays from the UK Consortium on Metabolic Phenotyping (MAP/UK).

Targeted metabolite assays that measure tens or hundreds of pre-selected metabolites, typically using liquid chromatography-mass spectrometry, are increasingly being developed and applied to metabolic phenotyping studies. These are used both as standalone phenotyping methods and for the validation of putative metabolic biomarkers obtained from untargeted metabolomics studies. However, there are no widely accepted standards in the scientific community for ensuring reliability of the development and validation of targeted metabolite assays (referred to here as 'targeted metabolomics'). Most current practices attempt to adopt, with modifications, the strict guidance provided by drug regulatory authorities for analytical methods designed largely for measuring drugs and other xenobiotic analytes. Here, the regulatory guidance provided by the European Medicines Agency, US Food and Drug Administration and International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use are summarized. In this Perspective, we have adapted these guidelines and propose a less onerous 'tiered' approach to evaluate the reliability of a wide range of metabolomics analyses, addressing the need for community-accepted, harmonized guidelines for tiers other than full validation. This 'fit-for-purpose' tiered approach comprises four levels-discovery, screening, qualification and validation-and is discussed in the context of a range of targeted and untargeted metabolomics assays. Issues arising with targeted multiplexed metabolomics assays, and how these might be addressed, are considered. Furthermore, guidance is provided to assist the community with selecting the appropriate degree of reliability for a series of well-defined applications of metabolomics.

Kuhuang alleviates liver fibrosis by modulating gut microbiota-mediated hepatic IFN signaling and bile acid synthesis.

Background: Liver fibrosis is a common outcome of the pathological progression of chronic liver disease; however, no specific and effective therapeutic agent has been approved for its treatment. We investigated the effects of Kuhuang on liver fibrosis and the underlying mechanisms of action. Materials and methods: To induce hepatic fibrosis, either 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet was administered, or bile duct ligation (BDL) surgery was performed on C57BL/6 mice. Kuhuang was orally administered to mice for 7days before and after bile duct ligation or 4weeks with a DDC diet. Hematoxylin and eosin, Sirius red staining, and immunohistochemical analyses were performed to evaluate hepatic pathology. Hepatic interferon-β (IFN-β) levels were measured using an enzyme-linked immunosorbent assay. RNA sequencing was performed to examine the gene expression profiles of liver tissues. The mRNA expression of inflammatory, profibrotic, and bile acid (BA)-related genes was further validated by qRT-PCR. A targeted metabolomics assay revealed the alteration of the hepatic bile acid (BA) composition. The composition of the gut microbiota was determined via 16S rRNA sequencing. Results: Treatment with Kuhuang attenuated liver fibrosis and reduced the inflammatory response in bile duct ligation and DDC mouse models. In addition, the hepatic IFN signaling pathway was activated following Kuhuang treatment. Kuhuang treatment also significantly decreased hepatic levels of both primary and secondary BAs. In addition, Kuhuang treatment altered gut microbiota composition, with an increased abundance of interferon-inducing Akkermansia and decreased abundance of bile salt hydrolase-producing Lactobacillus, Clostridium, and Bifidobacterium. Furthermore, the abundance of Akkermansia was positively correlated with the hepatic mRNA expression levels of Ifna4, Ifnb, and Isg15, whereas that of Lactobacillus, Clostridium - sensu - stricto - 1, and Bifidobacterium was positively correlated with levels of bile acid synthesis-related genes. Conclusion: Our results suggest that Kuhuang plays a protective role during the progression of liver fibrosis, potentially by altering the composition of the gut microbiota, which consequently activates interferon signaling and inhibits bile acid synthesis in the liver.

Diagnosing, discarding, or de-VUSsing: A practical guide to (un)targeted metabolomics as variant-transcending functional tests

For patients with inherited metabolic disorders (IMDs), any diagnostic delay should be avoided because early initiation of personalized treatment could prevent irreversible health damage. To improve diagnostic interpretation of genetic data, gene function tests can be valuable assets. For IMDs, variant-transcending functional tests are readily available through (un)targeted metabolomics assays. To support the application of metabolomics for this purpose, we developed a gene-based guide to select functional tests to either confirm or exclude an IMD diagnosis. Using information from a diagnostic IMD exome panel, Kyoto Encyclopedia of Genes and Genomes, and Inborn Errors of Metabolism Knowledgebase, we compiled a guide for metabolomics-based gene function tests. From our practical experience with this guide, we retrospectively selected illustrative cases for whom combined metabolomic/genomic testing improved diagnostic success and evaluated the effect hereof on clinical management. The guide contains 2047 metabolism-associated genes for which a validated or putative variant-transcending gene function test is available. We present 16 patients for whom metabolomic testing either confirmed or ruled out the presence of a second pathogenic variant, validated or ruled out pathogenicity of variants of uncertain significance, or identified a diagnosis initially missed by genetic analysis. Metabolomics-based gene function tests provide additional value in the diagnostic trajectory of patients with suspected IMD by enhancing and accelerating diagnostic success.

Abstract P3049: Age-independent Cardiac Protection By Pharmacological Activation Of Beclin-1 During Endotoxemia And Its Association With Energy Metabolic Reprogramming In Myocardium-A Targeted Metabolomics Study

Background: We previously showed that Beclin-1-dependent autophagy is cardiac protective in a rodent model of endotoxemia using young adult mice. In this report, we compared the potential therapeutic effects of pharmacological Beclin-1 activating peptide, TB-peptide, on the cardiac outcomes of young adult and aged mice during endotoxemia. We further examined alterations in myocardial metabolism induced by lipopolysaccharide (LPS) challenge with and without the TB-peptide treatment. Methods: C57BL/6J mice of 10-week and 24-month-old were challenged by LPS at doses at which cardiac dysfunction occurred. Following the treatment of TB-peptide or control vehicle, heart contractility, circulating cytokines, and myocardial autophagy were evaluated. A targeted metabolomics assay was applied to analyze cardiac metabolism. Results: TB-peptide boosted autophagic response, attenuated cytokine production, and improved cardiac performance in both young and aged mice during endotoxemia. A targeted metabolomics assay was designed to detect a pool of 361 known metabolites, of which 156 were detected in at least one of the heart tissue samples. LPS-induced impairments were found in glucose and amino acid (AA) metabolisms in mice of all ages, and TB-peptide provided ameliorative effects to rescue these alterations. However, lipid metabolites were upregulated in the young group but moderately downregulated in the aged by LPS, suggesting an age-dependent response. TB-peptide mitigated LPS-mediated trend of lipids in the young mice but provided little effect on the aged ones. Conclusion: Pharmacological activation of Beclin-1 by TB-peptide protects the heart in both young and aged population during endotoxemia, suggest a therapeutic potential for sepsis-induced cardiomyopathy. Metabolomics analysis suggests that this age-independent protection by TB-peptide is associated with reprograming of energy production via glucose and AA metabolisms.

Age-Independent Cardiac Protection by Pharmacological Activation of Beclin-1 During Endotoxemia and Its Association With Energy Metabolic Reprograming in Myocardium-A Targeted Metabolomics Study.

Background We showed that Beclin-1-dependent autophagy protects the heart in young and adult mice that underwent endotoxemia. Herein, we compared the potential therapeutic effects of Beclin-1 activating peptide, TB-peptide, on endotoxemia-induced cardiac outcomes in young adult and aged mice. We further evaluated lipopolysaccharide (lipopolysaccharide)-induced and TB-peptide treatment-mediated alterations in myocardial metabolism. Methods and Results C57BL/6J mice that were 10 weeks and 24 months old were challenged by lipopolysaccharide using doses at which cardiac dysfunction occurred. Following the treatment of TB-peptide or control vehicle, heart contractility, circulating cytokines, and myocardial autophagy were evaluated. We detected that TB-peptide boosted autophagy, attenuated cytokines, and improved cardiac performance in both young and aged mice during endotoxemia. A targeted metabolomics assay was designed to detect a pool of 361 known metabolites, of which 156 were detected in at least 1 of the heart tissue samples. Lipopolysaccharide-induced impairments were found in glucose and amino acid metabolisms in mice of all ages, and TB-peptide ameliorated these alterations. However, lipid metabolites were upregulated in the young group but moderately downregulated in the aged by lipopolysaccharide, suggesting an age-dependent response. TB-peptide mitigated lipopolysaccharide-mediated trend of lipids in the young mice but had little effect on the aged. (Study registration: Project DOI: https://doi.org/10.21228/M8K11W). Conclusions Pharmacological activation of Beclin-1 by TB-peptide is cardiac protective in both young and aged population during endotoxemia, suggest a therapeutic potential for sepsis-induced cardiomyopathy. Metabolomics analysis suggests that an age-independent protection by TB-peptide is associated with reprograming of energy production via glucose and amino acid metabolisms.

Ildr1 gene deletion protects against diet-induced obesity and hyperglycemia

ObjectiveImmunoglobulin-like Domain-Containing Receptor 1 (ILDR1) is expressed on nutrient sensing cholecystokinin-positive enteroendocrine cells of the gastrointestinal tract and it has the unique ability to induce fat-mediated CCK secretion. However, the role of ILDR1 in CCK-mediated regulation of satiety is unknown. In this study, we examined the effects of ILDR1 on food intake and metabolic activity using mice with genetically-deleted Ildr1.MethodsThe expression of ILDR1 in murine tissues and the measurement of adipocyte cell size were evaluated by light and fluorescence confocal microscopy. The effects of Ildr1 deletion on mouse metabolism were quantitated using CLAMS chambers and by targeted metabolomics assays of multiple tissues. Hormone levels were measured by ELISA. The effects of Ildr1 gene deletion on glucose and insulin levels were determined using in vivo oral glucose tolerance, meal tolerance, and insulin tolerance tests, as well as ex vivo islet perifusion.ResultsILDR1 is expressed in a wide range of tissues. Analysis of metabolic data revealed that although Ildr1-/- mice consumed more food than wild-type littermates, they gained less weight on a high fat diet and exhibited increased metabolic activity. Adipocytes in Ildr1-/- mice were significantly smaller than in wild-type mice fed either low or high fat diets. ILDR1 was expressed in both alpha and beta cells of pancreatic islets. Based on oral glucose and mixed meal tolerance tests, Ildr1-/- mice were more effective at lowering post-prandial glucose levels, had improved insulin sensitivity, and glucose-regulated insulin secretion was enhanced in mice lacking ILDR1.ConclusionIldr1 loss significantly modified metabolic activity in these mutant mice. While Ildr1 gene deletion increased high fat food intake, it reduced weight gain and improved glucose tolerance. These findings indicate that ILDR1 modulates metabolic responses to feeding in mice.

Associations Between Ambient Temperature and Circulating Maternal One-carbon and Amino Acid Metabolites in Pregnancy

ObjectivesExcessive ambient temperature closely linked to climate change is an imminent threat to the health of mothers and children globally. Recent studies have shown detrimental associations between high temperatures during pregnancy and birth outcomes. The goal of this study was to examine relationships between circulating metabolites in the mother and environmental temperature in a resource-limited setting. MethodsThe study was a secondary analysis employing data from the Women First (WF) trial conduced in Thatta, Pakistan. As part of the trial, women of child-bearing age either consumed a comprehensive maternal nutritional supplement (MNS) prior to conception until delivery (Arm 1), from 12 wk gestation until delivery (Arm 2); or were not supplemented with MNS Arm 3. Daily maximum air temperatures were acquired from the closest surface observation systems (n = 131). Average daily maximum temperatures (Tmax) for a 90-day window representing the 1st trimester of pregnancy was calculated for each participant. The number of days in this period when the Tmax was > 39°C was computed to assess ‘heat stress days’. Maternal blood was collected on dried blood spot (DBS) cards at 34 wk gestation. A targeted metabolomics assay measuring concentrations of 23 metabolites was performed on DBS samples using LC-MS/MS. ResultsLinear regression was employed to assess the associations between ambient temperature in the 1st trimester and metabolite levels (z-scores). Models were adjusted for MNS supplementation. Significant associations between Tmax in the 1st trimester and 7 metabolites were observed (FDR p-values < 0.05). Of these, maternal choline concentrations were negatively correlated with maximal temperature in the 1st trimester (β = –0.063, p = 4.3e-10). Concentrations of glutamine, histidine, arginine, symmetrical-dimethylarginine, methionine and cysteine were positively associated (p < 0.05) with daily maximal temperature. ConclusionsCollectively, the findings indicate that exposure to excessive heat stress in the first trimester is associated with lower choline levels and other metabolites important in blood pressure regulation. While the findings are associative, our results point to underlying mechanistic alterations in maternal circulation in concert with environmental heat stress. Funding SourcesBill & Melinda Gates Foundation; NIH/NICHD/ODS.

Osa-miR1320 targets the ERF transcription factor OsERF096 to regulate cold tolerance via JA-mediated signaling.

MicroRNAs play key roles in abiotic stress response. Rice (Oryza sativa L.) miR1320 is a species-specific miRNA that contributes to miR168-regulated immunity. However, it is still unknown whether miR1320 is involved in rice response to abiotic stress. In this study, we illustrated that the miR1320 precursor generated two mature miR1320s, miR1320-3p, and miR1320-5p, and they both displayed decreased expression under cold stress. Genetic evidence showed that miR1320 overexpression resulted in increased cold tolerance, while miR1320 knock down (KD) reduced cold tolerance. Furthermore, an APETALA2/ethylene-responsive factor (ERF) transcription factor OsERF096 was identified as a target of miR1320 via 5'-RACE and dual luciferase assays. OsERF096 expression was altered by miR1320 overexpression and KD and exhibited an opposite pattern to that of miR1320 in different tissues and under cold stress. Consistently, OsERF096 negatively regulated cold stress tolerance. Furthermore, we suggested that OsERF096 could bind to the GCC and DRE cis-elements and act as a transcriptional activator in the nucleus. Based on RNA-sequencing and targeted metabolomics assays, we found that OsERF096 modified hormone content and signaling pathways. Finally, phenotypic and reverse transcription-quantitative PCR assays showed that jasmonic acid (JA) methyl ester application recovered the cold-sensitive phenotype and JA-activated expression of three Dehydration Responsive Element Binding genes in the OsERF096-OE line. Taken together, our results strongly suggest that the miR1320-OsERF096 module regulates cold tolerance by repressing the JA-mediated cold signaling pathway.

A rapid LC-MS/MS assay for detection and monitoring of underivatized branched-chain amino acids in maple syrup urine disease

IntroductionQuantitation of the isomeric branched-chain amino acids (BCAA; valine, alloisoleucine, isoleucine, leucine) is a challenging task that typically requires derivatization steps or long runtimes if a traditional chromatographic method involving a ninhydrin ion pairing reagent is used. ObjectivesTo develop and perform clinical validation of a rapid, LC-MS/MS-based targeted metabolomics assay for detection and monitoring of underivatized BCAA in human plasma. Methods:Various columns and modes of chromatography were tested. The final optimized method utilized mixed mode chromatography with an Intrada column under isocratic condition. Sample preparation utilized the 96-well format. Briefly, extraction solvent containing the internal standard is added to 20 uL of sample, followed by shaking and positive pressure filtering, and the resulting extracted sample is analyzed. The assay was validated based on accepted quality standards (e.g., CLIA and CLSI) for clinical assays. ResultsThe method is linear over a wide range of concentrations, 2.0–1500 µM, with LOD of 0.60 µM and LOQ of 2.0 µM. The precision of the assay was 4–10% across analytes. The method was also validated against reference laboratories via blinded split-sample analysis and demonstrated good agreement with accuracy: 89–95% relative to the external group mean. ConclusionWe have developed a method that is accurate, rapid, and reliable for routine clinical testing of patient sample BCAA, which is used in the diagnosis and management of maple syrup urine disease (MSUD). The assay also has desirable characteristics, such as short run time, small sample volume requirement, simple sample preparation without the need for derivatization, and high throughput.

A Comprehensive Targeted Metabolomics Assay for Crop Plant Sample Analysis.

Metabolomics plays an important role in various fields from health to agriculture. However, the comprehensive quantitative metabolomic analysis of plants and plant metabolites has not been widely performed. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based plant metabolomics offers the sensitivity and breadth of coverage for both phenotyping and disease diagnosis of plants. Here, we report a high-coverage and quantitative MS-based assay for plant metabolite analysis. The assay detects and quantifies 206 primary and secondary plant metabolites, including many key plant hormones. In total, it measures 28 amino acids and derivatives, 27 organic acids, 20 biogenic amines and derivatives, 40 acylcarnitines, 90 phospholipids and C-6 sugars. All the analysis methods in this assay are based on LC-MS/MS techniques using both positive and negative-mode multiple reaction monitoring (MRM). The recovery rates of spiked plant samples at three different concentration levels (low, medium and high) ranged from 80% to 120%, with satisfactory precision values of less than 20%. This targeted plant metabolomic assay has been successfully applied to the analysis of large numbers of pine and spruce needle samples, canola root samples, as well as cannabis samples. Moreover, the assay was specifically developed in a 96-well plate format, which enables automated, high-throughput sample analysis. This assay has already been used to analyze over 1500 crop plant samples in less than two months.

ADP Induces Blood Glucose Through Direct and Indirect Mechanisms in Promotion of Hepatic Gluconeogenesis by Elevation of NADH.

Extracellular ADP, a derivative of ATP, interacts with the purinergic receptors in the cell membrane to regulate cellular activities. This signaling pathway remains unknown in the regulation of blood glucose in vivo. We investigated the acute activity of ADP in mice through a peritoneal injection. In the lean mice, in response to the ADP treatment, the blood glucose was elevated, and pyruvate tolerance was impaired. Hepatic gluconeogenesis was enhanced with elevated expression of glucogenic genes (G6pase and Pck1) in the liver. An elevation was observed in NADH, cAMP, AMP, GMP and citrate in the liver tissue in the targeted metabolomics assay. In the primary hepatocytes, ADP activated the cAMP/PKA/CREB signaling pathway, which was blocked by the antagonist (2211) of the ADP receptor P2Y13. In the circulation, gluconeogenic hormones including glucagon and corticosterone were elevated by ADP. Insulin and thyroid hormones (T3 and T4) were not altered in the blood. In the diet-induced obese (DIO) mice, NADH was elevated in the liver tissue to match the hepatic insulin resistance. Insulin resistance was intensified by ADP for further impairment in insulin tolerance. These data suggest that ADP induced the blood glucose through direct and indirect actions in liver. One of the potential pathways involves activation of the P2Y13/cAMP/PKA/CREB signaling pathway in hepatocytes and the indirect pathway may involve induction of the gluconeogenic hormones. NADH is a signal for gluconeogenesis in the liver of both DIO mice and lean mice.