Targeted metabolomics reveals the aberrant energy status in diabetic peripheral neuropathy and the neuroprotective mechanism of traditional Chinese medicine JinMaiTong

Diabetic peripheral neuropathy (DPN) is a common and devastating complication of diabetes, for which effective therapies are currently lacking. Disturbed energy status plays a crucial role in DPN pathogenesis. However, the integrated profile of energy metabolism, especially the central carbohydrate metabolism, remains unclear in DPN. Here, we developed a metabolomics approach by targeting 56 metabolites using high-performance ion chromatography-tandem mass spectrometry (HPIC-MS/MS) to illustrate the integrative characteristics of central carbohydrate metabolism in patients with DPN and streptozotocin-induced DPN rats. Furthermore, JinMaiTong (JMT), a traditional Chinese medicine (TCM) formula, was found to be effective for DPN, improving the peripheral neurological function and alleviating the neuropathology of DPN rats even after demyelination and axonal degeneration. JMT ameliorated DPN by regulating the aberrant energy balance and mitochondrial functions, including excessive glycolysis restoration, tricarboxylic acid cycle improvement, and increased adenosine triphosphate (ATP) generation. Bioenergetic profile was aberrant in cultured rat Schwann cells under high-glucose conditions, which was remarkably corrected by JMT treatment. In-vivo and in-vitro studies revealed that these effects of JMT were mainly attributed to the activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and downstream peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Our results expand the therapeutic framework for DPN and suggest the integrative modulation of energy metabolism using TCMs, such as JMT, as an effective strategy for its treatment.

Targeted metabolomics study of fatty-acid metabolism in lean metabolic-associated fatty liver disease patients

BACKGROUND The annual incidence of metabolic-associated fatty liver disease(MAFLD)in China has been increasing and is often overlooked owing to its insidious charac-teristics.Approximately 50%of the patients have a normal weight or are not obese.They are said to have lean-type MAFLD,and few studies of such patients are available.Because MAFLD is associated with abnormal lipid metabolism,lipid-targeted metabolomics was used in this study to provide experimental evidence for early diagnosis and pathogenesis.MAFLD and analyze metabolic pathways.UPLC-Q-Orbitrap/MS content determination was used to determine serum palmitic acid(PA),oleic acid(OA),linoleic acid(LA),and arachidonic acid(AA)levels in lean-type MAFLD patients.RESULTS Urea nitrogen and uric acid levels were higher in lean-type MAFLD patients than in healthy individuals(P<0.05).Alanine transaminase and cholinesterase levels were higher in lean-type MAFLD patients than in healthy indi-viduals(P<0.01).The expression of high-density lipoprotein and apolipoprotein A-1 were lower in lean-type MAFLD patients than in healthy individuals(P<0.05)and the expression of triglycerides and fasting blood glucose were increased(P<0.01).A total of 65 biomarkers that affected the synthesis and metabolism of fatty acids were found with P<0.05 and variable importance in projection>1.The levels of PA,OA,LA,and AA were significantly increased compared with healthy individuals.CONCLUSION The metabolic profiles of lean-type MAFLD patients and healthy participants differed significantly,yielding 65 identified biomarkers.PA,OA,LA,and AA exhibited the most significant changes,offering valuable clinical guidance for prevention and treatment of lean-type MAFLD.

Targeted metabolomics of sulfated bile acids in urine for the diagnosis and grading of intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy(ICP)is related to cholestatic disorder in pregnancy.Total urinary sulfated bile acids(SBAs)were found increased in ICP.We distinguished the metabolic profiling of urinary SBAs in ICP to find potential biomarkers for the diagnosis and grading of ICP.The targeted metabolomics based on high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)was used to analyze urinary SBAs profiling in mild and severe ICP cases,as well as healthy controls.16 kinds of urinary SBAs were determined by HPLC-MS/MS.Sulfated dihydroxy glycine bile acid(di-GBA-S),glycine cholic acid 3-sulfate(GCA-3S),sulfated dihydroxy taurine bile acid(di-TBA-S)and taurine cholic acid 3-sulfate(TCA-3S)increased significantly in ICP group compared with the control group.Seven kinds of SBAs were significantly different(p<0.05)between the ICP group and the control group,with the variable importance in the projection(VIP)value more than one by the orthogonal partial least squares discriminant analysis(OPLS-DA).GCA-3S was well-suited to be used as the biomarker for the diagnosis of ICP with the sensitivity of 100%and specificity of 95.5%.A multi-variable logistic regression containing GCA-3S and di-GBA-S-1 was constructed to distinguish severe ICP from mild ICP,with the sensitivity of 94.4%and specificity of 100%.The developed HPLC-MS/MS method is suitable for the measurement of urinary SBAs profiling.Moreover,the urinary SBAs in the metabolomic profiling have the potential to be used as non-intrusive biomarkers for the diagnosis and grading of ICP.

Effects of antibiotic antitumor drugs on nucleotide levels in cultured tumor cells:an exploratory method to distinguish the mechanisms of antitumor drug action based on targeted metabolomics

Nucleotide pools in mammalian cells change due to the influence of antitumor drugs,which may help in evaluating the drug effect and understanding the mechanism of drug action.In this study,an ion-pair RP-HPLC method was used for a simple,sensitive and simultaneous determination of the levels of 12 nucleotides in mammalian cells treated with antibiotic antitumor drugs(daunorubicin,epirubicin and dactinomycin D).Through the use of this targeted metabolomics approach to find potential biomarkers,UTP and ATP were verified to be the most appropriate biomarkers.Moreover,a holistic statistical approach was put forward to develop a model which could distinguish 4 categories of drugs with different mechanisms of action.This model can be further validated by evaluating drugs with different mechanismsof action.This targeted metabolomics study may provide a novel approach to predict the mechanism of action of antitumor drugs.

The role of the auxin-response genes MdGH3.1 and MdSAUR36 in bitter pit formation in apple

Apples often exhibit bitter pits in response to metabolic disorders during ripening and storage;however, the mechanisms underlying the bitter pit(BP) development remain unclear. Here, metabolome and transcriptome analyses were performed to investigate BP pulp of 'Fuji'. Two auxin-response genes, MdGH3.1 and MdSAUR36, were screened. Their expression as well as the auxin content in BP pulp were found to be higher than those in healthy pulp(P < 0.01). In the field, excess CO(NH2)2increased the incidence of BP. Moreover, the auxin content and MdGH3.1 expression increased in apples after nitrogen fertilization. On Day 30 before harvest, the two genes were transiently transferred to the fruit, and 20.69% and 23.21% of BP fruits were harvested. After 10 μmol·L-1auxin was infiltrated at low pressure into postharvest fruit, the increase in MdGH3.1 expression occurred earlier than that in MdSAUR36. MdGH3.1 increased the expression of MdSAUR36, but MdSAUR36 did not increase expression of MdGH3.1. Therefore, we suggest that MdGH3.1 acts upstream of MdSAUR36 during BP formation and that these genes induce BP formation by regulating auxin and phenylpropanoid biosynthesis.

EuSHT Acts as a Hub Gene Involved in the Biosynthesis of 6-Hydroxyluteolin and Quercetin Induced by Salt Stress in Eucommia ulmoides

Salt stress inhibits plant growth and affects the biosynthesis of its secondary metabolites.Flavonoids are natural compounds that possess many important biological activities,playing a significant role in the medicinal activity of Eucommia ulmoides(E.ulmoides).To investigate the mechanism by which salt stress affects the biosynthesis of flavonoids in E.ulmoides,a comprehensive analysis of metabolomics and transcriptomics was conducted.The results indicated that salt stress led to the wilting and darkening of E.ulmoides leaves,accompanied by a decrease in chlorophyll levels,and significantly induced malondialdehyde(MDA)and relative electrical conductivity.During salt stress,most metabolites in the flavonoid biosynthesis pathway of E.ulmoides were upregulated,indicating that flavonoid biosynthesis is likely the main induced pathway under salt stress.Among them,secondary metabolites such as 6-Hydroxyluteolin and Quercetin are likely key metabolites induced by salt stress.The correlation analysis of transcriptomics and metabolomics revealed that EuSHT is a hub gene induced by salt stress,promoting the production of flavonoids such as 6-Hydroxyluteolin.The co-expression network showed a strong positive correlation between EuSHT and the biosynthesis of 6-Hydroxyluteolin and Quercetin,while it exhibited a negative correlation with Catechin biosynthesis.The branches leading to Luteolin and Dihydroquercetin are likely the main pathways for flavonoid compound biosynthesis in the plant stress response during salt stress.The results of this study provided a preliminary mechanism of secondary metabolites such as flavonoids in the medicinal plant E.ulmoides induced by salt stress and provided new theoretical support for discussing the mechanism of plant stress response.It also provided useful information for subsequent exploration of resistance genes in E.ulmoides.

4-Hydroxy-2-Oxoglutaric Acid,A KeyMetabolite Involved in Trypsin-Regulation of Arginine Metabolism in Hylocereus undatus during Storage

Trypsin,a novel superoxide scavenger,significantly enhances the storage quality of Hylocereus undatus(H.undatus).To elucidate the preservation mechanism of trypsin on H.undatus,a widely targeted metabolomic analysis,and transcriptomics analysis were conducted.Firstly,a total of 453 metabolites were identified,with organic acids and their derivatives constituting the largest proportion(25%).Amino acids and their metabolites,prominent among organic acids,were further analyzed.Among them,73 metabolites were associated with amino acids,and 37 exhibited significant differences.The most enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway was arginine biosynthesis(map00220),with polyamine metabolites showing the most pronounced differences,particularly spermine(FC=1.7594).Compared with the control group,4-hydroxy-2-oxoglutaric acid was significantly upregulated(FC=2.117)in the process of spermine biosynthesis.Furthermore,the results of Gene Ontology(GO)and KEGG enrichment analysis of the H.undatus transcriptome profile revealed that trypsin treatment led to 187 differentially expressed genes associated with arginine.Both GO and KEGG analyses exhibited significant enrichment in the spermine biosynthetic process(GO:0006597)(map:00220)within the arginine biosynthesis pathway.Moreover,most enzymes and metabolites within the spermine biosynthesis pathway in H.undatus were upregulated.The results of the PPI network highlighted that ADC,SPDS,and SAMDC,among others,were pivotal proteins involved in trypsin-regulated arginine metabolism and spermine synthesis.This study revealed that trypsin could significantly delay postharvest senescence of H.undatus at room temperature.This effect might be attributed to trypsin triggering the synthesis of 4-hydroxy-2-oxoglutaric acid in the fruit peel,thereby promoting the biosynthesis of spermine and other polyamines.

Novel biomarkers identifying hypertrophic cardiomyopathy and its obstructive variant based on targeted amino acid metabolomics

Background:Hypertrophic cardiomyopathy(HCM)is an underdiagnosed genetic heart disease worldwide.The management and prognosis of obstructive HCM(HOCM)and non-obstructive HCM(HNCM)are quite different,but it also remains challenging to discriminate these two subtypes.HCM is characterized by dysmetabolism,and myocardial amino acid(AA)metabolism is robustly changed.The present study aimed to delineate plasma AA and derivatives profiles,and identify potential biomarkers for HCM.Methods:Plasma samples from 166 participants,including 57 cases of HOCM,52 cases of HNCM,and 57 normal controls(NCs),who first visited the International Cooperation Center for HCM,Xijing Hospital between December 2019 and September 2020,were collected and analyzed by high-performance liquid chromatography-mass spectrometry based on targeted AA metabolomics.Three separate classification algorithms,including random forest,support vector machine,and logistic regression,were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM.Results:The univariate analysis showed that the serine,glycine,proline,citrulline,glutamine,cystine,creatinine,cysteine,choline,and aminoadipic acid levels in the HCM group were significantly different from those in the NC group.Four AAs and derivatives(Panel A;proline,glycine,cysteine,and choline)were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs.The receiver operating characteristic(ROC)analysis in Panel A yielded an area under the ROC curve(AUC)of 0.83(0.75-0.91)in the training set and 0.79(0.65-0.94)in the validation set.Moreover,among 10 AAs and derivatives(arginine,phenylalanine,tyrosine,proline,alanine,asparagine,creatine,tryptophan,ornithine,and choline)with statistical significance between HOCM and HNCM,3 AAs(Panel B;arginine,proline,and ornithine)were selected to differentiate the two subgroups.The AUC values in the training and validation sets for Panel B were 0.83(0.74-0.93)and 0.82(0.66-0.98),respectively.Conclusions:The plasma AA and derivatives profiles were distinct between the HCM and NC groups.Based on the differential profiles,the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive.

Targeted metabolic analysis of nucleotides and identification of biomarkers associated with cancer in cultured cell models

Cancer,like other diseases accompanied by metabolic changes,shows characteristic DNA/RNA modifications and activities of modifying enzymes,resulting in fluctuations in nucleoside levels.In this study,we undertook targeted metabolomic analyses of nucleotides in different cancer cell culture models using a sensitive and reproducible ion-pair HPLC method.The experimental data were analyzed by principal component analysis(PCA)to identify potential biomarkers in cancer cells,and statistical significance was determined by one-way analysis of variance.As a result,a clear differentiation of normal and tumor cells into two clusters was shown,indicating abnormal metabolism of nucleotides in tumor cells.Six variables(AMP,UDP,CTP levels with a significance of Po0.05;ATP,UTP and GMP levels with a significance of Po0.01)were considered as potential biomarkers;the content of AMP,UTP,GMP and ATP was significantly higher in cancer cells.The receiver operating characteristic(ROC)curve analysis allowed us to discriminate normal cells from tumor cells based on area under the curve(AUC).The sequence of their AUC values were:ATP(0.979)4UTP(0.938)4CTP¼GMP(0.896)4AMP(0.812)4UDP(0.792),so we conclude that ATP and UTP are the best potential biomarkers in tumor cells.This study may provide a valuable tool for studying minute alterations of intracellular nucleotide pools induced by anticancer/antiviral drugs,diseases or environmental factors.

Integrated transcriptomics and metabolomics analyses reveal the molecular mechanisms of red-light on carotenoids biosynthesis in tomato fruit

Carotenoids are the main pigments responsible for the coloration and account for the major antioxidant activity of tomato(Solanum lycopersicum L.)fruit.Significant increments in total carotenoids and lycopene levels were observed in tomato fruit illuminated by red light relative to white light in previous studies,but the mechanism of carotenoid biosynthesis regulated by red light is stll unclear.In the present study,the influence of red light on carotenoid biosynthesis in postharvest tomato fruit was conducted using targeted metabolomics and transcriptomic methods.A total of 25 differentially accumulated carotenoids and 1939 differentially expressed genes were isolated and identified.The results illustrated that the contents of phytoene and lycopene were considerably higher in fruit treated with red light than those with white light at 12 h.These differentially expressed genes are mainly enriched in plant hormone signal transduction,photosynthesis,secondary metabolite biosynthesis,and plant circadian rhythm.Moreover,from the results of coexpression network analysis,15 transcription factors from red light-treated fruit were screened;among these,transcription factors of SIERF4,SlbHLH93 and SllAA29,which are involved in signal transduction of light and hormones,respectively,may also play important roles in carotenoid biosynthesis regulated by red light in tomato fruit.It is concluded that red light enhanced carotenoid biosynthesis in postharvest tomato fruit and the mechanisms of enhanced carotenoid biosynthesis were not only associated with the direct regulation by red light signalling,but also with the indirect regulation by hormonal signalling.

Aerial ammonia exposure induces the perturbation of the interorgan ammonia disposal and branched-chain amino acid catabolism in growing pigs

Aerial ammonia exposure leads to tissue damage and metabolic dysfunction.However,it is unclear how different organs are coordinated to defend against aerial ammonia exposure.Twenty-four pigs were randomly divided into 4 groups,exposed to 0,10,25 or 35 mg/m^(3) ammonia respectively for 25 d.After above 25 mg/m^(3) ammonia exposure,decreased aspartate(P=0.016),glutamate(P=0.030)and increased ornithine(P=0.002)were found in the ammonia-removing liver,and after high ammonia(35 mg/m^(3))exposure,glutamine synthetase(GS)expression was increased(P=0.012).An increased glutamate(P=0.004)and decreased glutaminase(GLS)expression(P=0.083)were observed in the lungs after high ammonia exposure.There was also an increasing trend of glutamine in the kidneys after high ammonia exposure(P=0.066).For branched-chain amino acid(BCAA)catabolism,high ammonia exposure increased BCAA content in both the lungs and muscle(P<0.05),whereas below 25 mg/m^(3) ammonia exposure increased BCAA only in the lungs(P<0.05).The expression of BCAA transaminase(BCAT1/2)and dehydrogenase complex(BCKDHA/B and DBT)were inhibited to a varying degree in the liver,lungs and muscle after above 25 mg/m^(3) ammonia exposure,especially high ammonia exposure.The expression of BCKDH complex and glutamate-glutamine metabolism-related genes were highly expressed in the liver,followed by the lungs and muscle(P<0.01),whereas the BCAT2 expression was highest in the lungs(P=0.002).Altogether,low ammonia exposure sufficed to evoke the urea cycle to detoxify ammonia in the liver.The process of ammonia removal in the liver and potential ability of the lungs to detoxify ammonia were enhanced with increasing ammonia.Furthermore,high ammonia exposure impaired the BCAA catabolism and decreased the transcripts of the BCAA catabolism-related enzymes,resulting in high BCAA content in extrahepatic tissues.Therefore,with aerial ammonia increasing,an increased urea cycle and glutamine synthesis were ammonia defensive strategies,and high ammonia exposure impaired the BCAA catabolism.

Combinatorial analysis of transcription and metabolism reveals the regulatory network associated with antioxidant substances in waxy corn

Maize is an essential source of nutrition for humans and animals and is rich in various metabolites that determine its quality.Different maize varieties show significant differences in metabolite content.Two kinds of waxy maize parental materials,S181 and 49B,created by the Chongqing Academy of Agricultural Sciences,are widely grown in China.S181 shows higher starch and sugar contents than 49B.This study generated metabolic profiles to assess the differences between the two varieties.A total of 674 metabolites that were significantly differentially expressed between the two varieties were identified by gas chromatography and untargeted metabolomics technology.These metabolites were associated with 21 categories,including antioxidant metabolites.Moreover,6415 differentially expressed genes(DEGs)were identified by RNA-seq.Interestingly,these DEGs comprised starch and sugar synthesis pathway genes and 72 different transcription factor families.Among these,six families that were reported to play an essential role in plant antioxidant action accounted for 39.2%of the transcription factor families.Using the Kyoto Encyclopedia of Genes and Genomes(KEGG)classification,the DEGs were mainly involved in amino acid biosynthesis,glycolysis/glucose metabolism,and the synthetic and metabolic pathways of antioxidant active substances.Furthermore,the correlation analysis of transcriptome and metabolomics identified five key transcription factors(ZmbHLH172,ZmNAC44,ZmNAC-like18,ZmS1FA2,ZmERF172),one ubiquitin ligase gene(ZmE25A)and one sucrose synthase gene(ZmSS1).They likely contribute to the quality traits of waxy corn through involvement in the metabolic regulatory network of antioxidant substances.Thus,our results provide new insights into maize quality-related antioxidant metabolite networks and have potential applications for waxy corn breeding.

Association of altered serum acylcarnitine levels in early pregnancy and risk of gestational diabetes mellitus

Gestational diabetes mellitus(GDM)is a high-prevalence disease and diagnosed in middle pregnancy.Acylcarnitines are a series of fatty acid esters of carnitine and play important roles in fatty acid and carbohydrate metabolism.However,the role of acylcarnitine on the development of GDM remains unclear.This case-control study involving 214 study participants(107 GDM cases and 107 matched controls)was conducted in a cohort,in China,from 2013 to 2015.The levels of carnitine and 36 acylcarnitines in serum samples collected at the early stage of pregnancy were determined by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry.The associations of the levels of the 37 targeted compounds with GDM risk were investigated by using binary conditional logistic regression models.Alterations in acylcarnitine levels were observed 9–17 weeks before GDM diagnosis.The increases in levels of propionyl-carnitine,malonyl-carnitine,isovaleryl-carnitine,palmitoyl-carnitine and linoleoyl-carnitine were associated with GDM risk with odds ratios(ORs)per standard deviation(SD)increment greater than 1(p<0.05),after adjustment for potential confounding factors(pre-pregnancy body mass index and parity).On the contrary,the increases of decanoyl-carnitine,decenoyl-carnitine,tetradecenoyl-carnitine,tetradecandienoylcarnitine levels were associated with the reduced risk for GDM(ORs per SD<1,p<0.05).To our knowledge,the present study is the largest case-control study to investigate the association between early-pregnancy acylcarnitine levels in serum and GDM risk.The findings add to the evidence for the association between acylcarnitine levels and GDM risk.