FOXA3 regulates cholesterol metabolism to compensate for low uptake during the progression of lung adenocarcinoma.

Cholesterol metabolism is vital for multiple cancer progression, while how cholesterol affects lung, a low-cholesterol tissue, for cancer metastasis and the underlying mechanism remain unclear. In this study, we found that metastatic lung adenocarcinoma cells acquire cellular dehydrocholesterol and cholesterol by endogenous cholesterol biosynthesis, instead of uptake upon cholesterol treatment. Besides, we demonstrated that exogenous cholesterol functions as signaling molecule to induce FOXA3, a key transcription factor for lipid metabolism via GLI2. Subsequently, ChIP-seq analysis and molecular studies revealed that FOXA3 transcriptionally activated Hmgcs1, an essential enzyme of cholesterol biosynthesis, to induce endogenous dehydrocholesterol and cholesterol level for membrane composition change and cell migration. Conversely, FOXA3 knockdown or knockout blocked cholesterol biosynthesis and lung adenocarcinoma metastasis in mice. In addition, the potent FOXA3 inhibitor magnolol suppressed metastatic gene programs in lung adenocarcinoma patient-derived organoids (PDOs). Altogether, our findings shed light onto unique cholesterol metabolism and FOXA3 contribution to lung adenocarcinoma metastasis.

Complete genomic analysis of rabbit rotavirus G3P[22] in China.

A rabbit rotavirus Z3171 isolate from diarrheic rabbits was identified and sequenced. The genotype constellation of Z3171 is G3-P[22]-I2-R3-C3-M3-A9-N2-T1-E3-H3, which is different from the constellation observed in previously characterized LRV strains. However, the genome of Z3171 differed substantially from those of the rabbit rotavirus strains N5 and Rab1404 in terms of both gene content and gene sequence. Our study suggests that either a reassortment event occurred between human and rabbit rotavirus strains or there are undetected genotypes circulating in the rabbit population. This is the first report of detection of a G3P[22] RVA strain in rabbits in China.

Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus.

Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as 94.4 and 99.2%, respectively, with the strong agreement (κ -0.58) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.

In this study, we used a Chinese porcine rotavirus-A (PoRVA) strain containing the I5, a dominant VP6-genotype in pigs, for production of VP6 (most conserved) protein based polyclonal antibodies (pAb) in rabbits (as capture Ab) and mouse (as detector Ab) for development of simple, cost effective, highly specific and sensitive AC-ELISA for detection of PoRVA. Furthermore, there is no any previous published report on application of rabbit and mouse pAb against VP6 for developing an AC-ELISA against PoRVA.

Dephosphorylation of AMP-activated protein kinase exacerbates ischemia/reperfusion-induced acute kidney injury via mitochondrial dysfunction.

Kidney tubular epithelial cells are high energy-consuming epithelial cells that depend mainly on fatty acid oxidation for an energy supply. AMP-activated protein kinase (AMPK) is a key regulator of energy production in most cells, but the function of AMPK in tubular epithelial cells in acute kidney disease is unclear. Here, we found a rapid decrease in Thr172-AMPKα phosphorylation after ischemia/reperfusion in both in vivo and in vitro models. Mice with kidney tubular epithelial cell-specific AMPKα deletion exhibited exacerbated kidney impairment and apoptosis of tubular epithelial cells after ischemia/reperfusion. AMPKα deficiency was accompanied by the accumulation of lipid droplets in the kidney tubules and the elevation of ceramides and free fatty acid levels following ischemia/reperfusion injury. Mechanistically, ischemia/reperfusion triggered ceramide production and activated protein phosphatase PP2A, which dephosphorylated Thr172-AMPKα. Decreased AMPK activity repressed serine/threonine kinase ULK1-mediated autophagy and impeded clearance of the dysfunctional mitochondria. Targeting the PP2A-AMPK axis by the allosteric AMPK activator C24 restored fatty acid oxidation and reduced tubular cell apoptosis during ischemia/reperfusion-induced injury, by antagonizing PP2A dephosphorylation and promoting the mitophagy process. Thus, our study reveals that AMPKα plays an important role in protecting against tubular epithelial cell injury in ischemia/reperfusion-induced acute kidney injury. Hence, activation of AMPK could be a potential therapeutic strategy for acute kidney injury treatment.

Loss of SIRT5 promotes bile acid-induced immunosuppressive microenvironment and hepatocarcinogenesis.

BACKGROUND & AIMS: The liver is a metabolically active organ and is also 'tolerogenic', exhibiting sophisticated mechanisms of immune regulation that prevent pathogen attacks and tumorigenesis. How metabolism impacts the tumor microenvironment (TME) in hepatocellular carcinoma (HCC) remains understudied. METHODS: We investigated the role of the metabolic regulator SIRT5 in HCC development by conducting metabolomic analysis, gene expression profiling, flow cytometry and immunohistochemistry analyses in oncogene-induced HCC mouse models and human HCC samples. RESULTS: We show that SIRT5 is downregulated in human primary HCC samples and that Sirt5 deficiency in mice synergizes with oncogenes to increase bile acid (BA) production, via hypersuccinylation and increased BA biosynthesis in the peroxisomes of hepatocytes. BAs act as a signaling mediator to stimulate their nuclear receptor and promote M2-like macrophage polarization, creating an immunosuppressive TME that favors tumor-initiating cells (TICs). Accordingly, high serum levels of taurocholic acid correlate with low SIRT5 expression and increased M2-like tumor-associated macrophages (TAMs) in HCC patient samples. Finally, administration of cholestyramine, a BA sequestrant and FDA-approved medication for hyperlipemia, reverses the effect of Sirt5 deficiency in promoting M2-like polarized TAMs and liver tumor growth. CONCLUSIONS: This study uncovers a novel function of SIRT5 in orchestrating BA metabolism to prevent tumor immune evasion and suppress HCC development. Our results also suggest a potential strategy of using clinically proven BA sequestrants for the treatment of patients with HCC, especially those with decreased SIRT5 and abnormally high BAs. LAY SUMMARY: Hepatocellular caricinoma (HCC) development is closely linked to metabolic dysregulation and an altered tumor microenvironment. Herein, we show that loss of the metabolic regulator Sirt5 promotes hepatocarcinogenesis, which is associated with abnormally elevated bile acids and subsequently an immunosuppressive microenvironment that favors HCC development. Targeting this mechanism could be a promising clinical strategy for HCC.

Myelocytomatosis-Protein Arginine N-Methyltransferase 5 Axis Defines the Tumorigenesis and Immune Response in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: HCC is a leading cause of cancer-related deaths globally with poor outcome and limited therapeutic options. Although the myelocytomatosis (MYC) oncogene is frequently dysregulated in HCC, it is thought to be undruggable. Thus, the current study aimed to identify the critical downstream metabolic network of MYC and develop therapies for MYC-driven HCC. APPROACH AND RESULTS: Liver cancer was induced in mice with hepatocyte-specific disruption of Myc and control mice by administration of diethylnitrosamine. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses revealed that urinary dimethylarginine, especially symmetric dimethylarginine (SDMA), was increased in the HCC mouse model in an MYC-dependent manner. Analyses of human samples demonstrated a similar induction of SDMA in the urines from patients with HCC. Mechanistically, Prmt5, encoding protein arginine N-methyltransferase 5, which catalyzes SDMA formation from arginine, was highly induced in HCC and identified as a direct MYC target gene. Moreover, GSK3326595, a PRMT5 inhibitor, suppressed the growth of liver tumors in human MYC-overexpressing transgenic mice that spontaneously develop HCC. Inhibition of PRMT5 exhibited antiproliferative activity through up-regulation of the tumor suppressor gene Cdkn1b/p27, encoding cyclin-dependent kinase inhibitor 1B. In addition, GSK3326595 induced lymphocyte infiltration and major histocompatibility complex class II expression, which might contribute to the enhanced antitumor immune response. Combination of GSK3326595 with anti-programed cell death protein 1 (PD-1) immune checkpoint therapy (ICT) improved therapeutic efficacy in HCC. CONCLUSIONS: This study reveals that PRMT5 is an epigenetic executer of MYC, leading to repression of the transcriptional regulation of downstream genes that promote hepatocellular carcinogenesis, highlights a mechanism-based therapeutic strategy for MYC-driven HCC by PRMT5 inhibition through synergistically suppressed proliferation and enhanced antitumor immunity, and finally provides an opportunity to mitigate the resistance of "immune-cold" tumor to ICT.

Bile acid and receptors: biology and drug discovery for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD), a series of liver metabolic disorders manifested by lipid accumulation within hepatocytes, has become the primary cause of chronic liver diseases worldwide. About 20%-30% of NAFLD patients advance to nonalcoholic steatohepatitis (NASH), along with cell death, inflammation response and fibrogenesis. The pathogenesis of NASH is complex and its development is strongly related to multiple metabolic disorders (e.g. obesity, type 2 diabetes and cardiovascular diseases). The clinical outcomes include liver failure and hepatocellular cancer. There is no FDA-approved NASH drug so far, and thus effective therapeutics are urgently needed. Bile acids are synthesized in hepatocytes, transported into the intestine, metabolized by gut bacteria and recirculated back to the liver by the enterohepatic system. They exert pleiotropic roles in the absorption of fats and regulation of metabolism. Studies on the relevance of bile acid disturbance with NASH render it as an etiological factor in NASH pathogenesis. Recent findings on the functional identification of bile acid receptors have led to a further understanding of the pathophysiology of NASH such as metabolic dysregulation and inflammation, and bile acid receptors are recognized as attractive targets for NASH treatment. In this review, we summarize the current knowledge on the role of bile acids and the receptors in the development of NAFLD and NASH, especially the functions of farnesoid X receptor (FXR) in different tissues including liver and intestine. The progress in the development of bile acid and its receptors-based drugs for the treatment of NASH including bile acid analogs and non-bile acid modulators on bile acid metabolism is also discussed.

In Vivo Efficacy and Metabolism of the Antimalarial Cycleanine and Improved In Vitro Antiplasmodial Activity of Semisynthetic Analogues.

Bisbenzylisoquinoline (BBIQ) alkaloids are a diverse group of natural products that demonstrate a range of biological activities. In this study, the in vitro antiplasmodial activity of three BBIQ alkaloids (cycleanine [compound 1], isochondodendrine [compound 2], and 2'-norcocsuline [compound 3]) isolated from the Triclisia subcordata Oliv. medicinal plant traditionally used for the treatment of malaria in Nigeria are studied alongside two semisynthetic analogues (compounds 4 and 5) of cycleanine. The antiproliferative effects against a chloroquine-resistant Plasmodium falciparum strain were determined using a SYBR green 1 fluorescence assay. The in vivo antimalarial activity of cycleanine is then investigated in suppressive, prophylactic, and curative murine malaria models after infection with a chloroquine-sensitive Plasmodium berghei strain. BBIQ alkaloids (compounds 1 to 5) exerted in vitro antiplasmodial activities with 50% inhibitory concentration (IC50) at low micromolar concentrations and the two semisynthetic cycleanine analogues showed an improved potency and selectivity compared to those of cycleanine. At oral doses of 25 and 50 mg/kg body weight of infected mice, cycleanine suppressed the levels of parasitemia and increased mean survival times significantly compared to those of the control groups. The metabolites and metabolic pathways of cycleanine were also studied using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. Twelve novel metabolites were detected in rats after intragastric administration of cycleanine. The metabolic pathways of cycleanine were demonstrated to involve hydroxylation, dehydrogenation, and demethylation. Overall, these in vitro and in vivo results provide a basis for the future evaluation of cycleanine and its analogues as leads for further development.

Antiviral activity of Piscidin 1 against pseudorabies virus both in vitro and in vivo.

BACKGROUND: Swine-origin virus infection spreading widely could cause significant economic loss to porcine industry. Novel antiviral agents need to be developed to control this situation. METHODS: In this study, we evaluated the activities of five broad-spectrum antimicrobial peptides (AMPs) against several important swine-origin pathogenic viruses by TCID50 assay. Plaque reduction assay and cell apoptosis assay were also used to test the activity of the peptides. Protection effect of piscidin against pseudorabies virus (PRV) was also examined in mouse model. RESULTS: Piscidin (piscidin 1), caerin (caerin 1.1) and maculatin (maculatin 1.1) could inhibit PRV by direct interaction with the virus particles in a dose-dependent manner and they could also protect the cells from PRV-induced apoptosis. Among the peptides tested, piscidin showed the strongest activity against PRV. Moreover, in vivo assay showed that piscidin can reduce the mortality of mice infected with PRV. CONCLUSION: In vitro and in vivo experiments indicate that piscidin has antiviral activity against PRV.

Proteome analysis of reticuloendotheliosis-virus-infected chicken embryo fibroblast cells through iTRAQ-based quantitative proteomics.

Reticuloendotheliosis virus (REV) is an important representative avian retrovirus. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of REV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect changes in protein levels in chicken embryo fibroblast cells (CEFs) that were infected with REV or mock infected. In total, 605 cellular proteins were differentially expressed, among which 196, 345, and 286 were differentially expressed in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. Gene Ontology analysis indicated that the biological processes of the differentially expressed proteins were primarily related to cellular processes, metabolic processes, biological regulation, response to stimulus, and immune system processes and that the molecular functions in which the differentially expressed proteins were mainly involved were binding, catalytic activity, and enzyme regulator activity. Pathway analysis showed that a total of 143, 167, and 179 pathways, including protein digestion and absorption, focal adhesion, ECM-receptor interaction, cytokine-cytokine receptor interaction, Toll-like receptors, and JAK-STAT signaling, were enriched in REV-infected CEFs at 1, 3, and 5 days postinfection, respectively. In conclusion, this study is the first to analyze the protein profile of REV-infected CEFs using an iTRAQ approach. The results of this study provide valuable information for better understanding the host response to REV infection.

A sensitive HPLC-MS/MS method for the simultaneous determination of anemoside B4, anemoside A3 and 23-hydroxybetulinic acid: Application to the pharmacokinetics and liver distribution of Pulsatilla chinensis saponins.

Pulsatilla chinensis saponins, the major active components in the herb, have drawn great attention as potential hepatitis B virus infection and hepatoma treatments. Here, a sensitive and accurate HPLC-MS/MS method was established for simultaneous determination of three saponins - anemoside B4, anemoside A3 and 23-hydroxybetulinic acid - in rat plasma and liver, and fully validated. The method was successfully applied to a pharmacokinetics and liver distribution study of P. chinensis saponins. Consequently, 23-hydroxybetulinic acid, with an extremely low content in the P. chinensis saponins, exhibited the highest exposure in the liver and in sites before and after hepatic disposition, namely, in the portal vein plasma and systemic plasma, followed by anemoside B4, which showed the highest content in the herb, whereas anemoside A3 displayed quite limited exposure. The hepatic first-pass effects were 71% for 23-hydroxybetulinic acid, 27% for anemoside B4 and 37% for anemoside A3, corresponding to their different extents of liver distribution. To our knowledge, this is the first investigation on the liver first-pass effect and distribution of P. chinensis saponins to date. These results also provide valuable information for the understanding of the pharmacological effect of P. chinensis saponins on liver diseases.

Determination and removal of sulfonamides and quinolones from environmental water samples using magnetic adsorbents.

In this study, the magnetic materials known as polymerized ionic liquid@3-(trimethoxysilyl)propyl methacrylate@Fe3 O4 nanoparticles were synthesized and utilized as potential adsorbents. First, these nanoparticles were applied to the analysis of sulfonamides and quinolones present in different water samples using magnetic solid phase extraction and high-performance liquid chromatography. Under optimized conditions, the developed method showed excellent detection sensitivity, with limits of detection (S/N = 3) and quantification limits (S/N = 10) within 0.2-1.0 and 0.8-3.4 µg/L, respectively. The spiked recoveries of the SAs and QNs in environmental water samples ranged from 83.5 to 103.0%, with RSDs of less than 4.5%. In addition, the adsorbents effectively removed sulfamethoxazole and ofloxacin present in existing aquatic environments. The adsorption kinetics and isotherms of sulfamethoxazole and ofloxacin on the magnetic adsorbents were studied to assess removal performance. The results indicate that the adsorption process follows a pseudo-second-order mechanism, which reveals that the sorption mechanism is the rate-limiting step and produces high qmax values (sulfamethoxazole = 70.35 mg/g and ofloxacin = 48.95 mg/g), thus demonstrating the enormous adsorption capacity of these magnetic adsorbents.

Porcine epidemic diarrhea virus ORF3 gene prolongs S-phase, facilitates formation of vesicles and promotes the proliferation of attenuated PEDV.

Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that has received increasing attention since the emergence of a PEDV variant worldwide. Previous studies have shown that PEDV ORF3 encodes an ion channel protein. However, its influence on cell cycle and subcellular structure still require more research. In this study, we developed a Vero cell line that stably expresses PEDV ORF3 gene. Subcellular localization and influences of PEDV ORF3 on host cells were investigated. We further verified whether or not this gene enhances virus production. The results showed that PEDV ORF3 protein localizes in the cytoplasm and affects cell cycle progression by prolonging the S phase. In addition, the ORF3-expressing Vero cells had more vesicles than the host Vero cells. Furthermore, the attenuated PEDV rather than virulent PEDV could grow better in ORF3-expressing Vero cells. The expression level of the PEDV nucleocapsid protein also increased. These results provided information on the function of PEDV ORF3 and were helpful in understanding the mechanisms of PEDV replication.

Sevoflurane Alters Serum Metabolites in Elders and Aging Mice and Increases Inflammation in Hippocampus.

Purpose: Postoperative cognitive dysfunction (POCD) is a central nervous system complication that occurs after anesthesia, particularly among the elderly. However, the neurological pathogenesis of postoperative cognitive dysfunction remains unclear. The aim of this study was to evaluate the effects of sevoflurane exposure on serum metabolites and hippocampal gene expression in elderly patients and aging mice by metabolomics and transcriptomic analysis and to explore the pathogenesis of sevoflurane induced POCD. Patients and Methods: Human serum samples from five patients over 60 years old were collected before sevoflurane anesthesia and 1 hour after anesthesia. Besides, mice aged at 12 months (n=6 per group) were anesthetized with sevoflurane for 2 hours or with sham procedure. Subsequently, serum and hippocampal tissues were harvested for analysis. Further investigation into the relationship between isatin and neuroinflammation was conducted using BV2 microglial cells. Results: Sevoflurane anesthesia led to the activation of inflammatory pathways, an increased presence of hippocampal astrocytes and microglia, and elevated expression of neuroinflammatory cytokines. Comparative analysis identified 12 differential metabolites that exhibited changes in both human and mouse serum post-sevoflurane anesthesia. Notably, isatin levels were significantly decreased after anesthesia. Notably, isatin levels significantly decreased after anesthesia, a factor known to stimulate proliferation and proinflammatory gene expression in microglia-the pivotal cell type in inflammatory responses. Conclusion: Sevoflurane-induced alterations in serum metabolites in both elderly patients and aging mice, subsequently contributing to increased inflammation in the hippocampus.

Obesity induced disruption on diurnal rhythm of insulin sensitivity via gut microbiome-bile acid metabolism.

The disruption of the diurnal rhythm has been recognized as a significant contributing factor to metabolic dysregulation. The important role of gut microbiota and bile acid metabolism has attracted extensive attention. However, the function of the gut microbiota-bile acid axis in regulating the diurnal rhythms of metabolic homeostasis remains largely unknown. Herein, we aimed to investigate the interplay between rhythmicity of host metabolism and gut microbiota-bile acid axis, as well as to assess the impact of obesity on them. We found that high fat diet feeding and Leptin gene deficiency (ob/ob) significantly disturbed the rhythmic patterns of insulin sensitivity and serum total cholesterol levels. The bile acid profiling unveiled a conspicuous diurnal rhythm oscillation of ursodeoxycholic acid (UDCA) in lean mice, concomitant with fluctuations in insulin sensitivity, whereas it was absent in obese mice. The aforementioned diurnal rhythm oscillations were largely desynchronized by gut microbiota depletion, suggesting the indispensable role of gut microbiota in diurnal regulation of insulin sensitivity and bile acid metabolism. Consistently, 16S rRNA sequencing revealed that UDCA-associated bacteria exhibited diurnal rhythm oscillations that paralleled the fluctuation in insulin sensitivity. Collectively, the current study provides compelling evidence regarding the association between diurnal rhythm of insulin sensitivity and gut microbiota-bile acid axis. Moreover, we have elucidated the deleterious effects of obesity on gut microbiome-bile acid metabolism in both the genetic obesity model and the diet-induced obesity model.

Xanthine dehydrogenase rewires metabolism and the survival of nutrient deprived lung adenocarcinoma cells by facilitating UPR and autophagic degradation.

Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid. The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown. Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis. Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells. Similar results were obtained in XDH-knockout cells. Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells. Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells. Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells. Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling. Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH. Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo. In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.

Development of a simultaneous quantification method for the gut microbiota-derived core nutrient metabolome in mice and its application in studying host-microbiota interaction.

The gut microbiota interacts with the host via production of various metabolites of dietary nutrients. Herein, we proposed the concept of the gut microbiota-derived core nutrient metabolome, which covers 43 metabolites in carbohydrate metabolism, glycolysis, tricarboxylic acid cycle and amino acid metabolism, and established a quantitative UPLC-Q/TOF-MS method through 3-nitrophenylhydrazine derivatization to investigate the influence of obesity on the gut microbiota in mice. All metabolites could be simultaneously analyzed via separation on a BEH C18 column within 18 min. The lower limits of quantification of most analytes were less than 1 µM. Validation results demonstrated suitability for the analysis of mouse fecal samples. The method was then applied to detect the gut microbiota-derived nutrient metabolome in the feces of high-fat diet induced obese (DIO) and ob/ob (leptin-deficient) mice, as well as obesity-prone (OP) and obesity-resistant (OR) mice. Compared to the control groups, there were 13, 23 and 10 differentially abundant metabolites detected in ob/ob, DIO and OP groups, respectively. Among them, amino acids including leucine, isoleucine, glycine, methionine, tyrosine and glutamine were co-downregulated in the obese or OP mice and exhibited inverse association with body weight. 16S rDNA analysis revealed that the genera Lactobacillus and Dubosiella were also inversely associated with body weight and positively correlated with fecal amino acids. Collectively, our work provides an effective and simplified method for simultaneous quantifying the gut microbiota-derived core nutrient metabolome in mouse feces, which could assist various future studies on host-microbiota metabolic interaction.

An integrative profiling of metabolome and transcriptome in the plasma and skeletal muscle following an exercise intervention in diet-induced obese mice.

Exercise intervention at the early stage of type 2 diabetes mellitus (T2DM) can aid in the maintenance of blood glucose homeostasis and prevent the development of macrovascular and microvascular complications. However, the exercise-regulated pathways that prevent the development of T2DM remain largely unclear. In this study, two forms of exercise intervention, treadmill training and voluntary wheel running, were conducted for high-fat diet (HFD)-induced obese mice. We observed that both forms of exercise intervention alleviated HFD-induced insulin resistance and glucose intolerance. Skeletal muscle is recognized as the primary site for postprandial glucose uptake and for responsive alteration beyond exercise training. Metabolomic profiling of the plasma and skeletal muscle in Chow, HFD, and HFD-exercise groups revealed robust alterations in metabolic pathways by exercise intervention in both cases. Overlapping analysis identified nine metabolites, including beta-alanine, leucine, valine, and tryptophan, which were reversed by exercise treatment in both the plasma and skeletal muscle. Transcriptomic analysis of gene expression profiles in the skeletal muscle revealed several key pathways involved in the beneficial effects of exercise on metabolic homeostasis. In addition, integrative transcriptomic and metabolomic analyses uncovered strong correlations between the concentrations of bioactive metabolites and the expression levels of genes involved in energy metabolism, insulin sensitivity, and immune response in the skeletal muscle. This work established two models of exercise intervention in obese mice and provided mechanistic insights into the beneficial effects of exercise intervention on systemic energy homeostasis.

Glutamine Production by Glul Promotes Thermogenic Adipocyte Differentiation Through Prdm9-Mediated H3K4me3 and Transcriptional Reprogramming.

Thermogenic adipocytes have been extensively investigated because of their energy-dissipating property and therapeutic potential for obesity and diabetes. Besides serving as fuel sources, accumulating evidence suggests that intermediate metabolites play critical roles in multiple biological processes. However, their role in adipocyte differentiation and thermogenesis remains unexplored. Here, we report that human and mouse obesity is associated with marked downregulation of glutamine synthetase (Glul) expression and activity in thermogenic adipose tissues. Glul is robustly upregulated during brown adipocyte (BAC) differentiation and in brown adipose tissue (BAT) upon cold exposure and Cl316,243 stimulation. Further genetic, pharmacologic, or metabolic manipulations of Glul and glutamine levels reveal that glutamine cells autonomously stimulate BAC differentiation and function and BAT remodeling and improve systemic energy homeostasis in mice. Mechanistically, glutamine promotes transcriptional induction of adipogenic and thermogenic gene programs through histone modification-mediated chromatin remodeling. Among all the glutamine-regulated writer and eraser genes responsible for histone methylation and acetylation, only Prdm9, a histone lysine methyltransferase, is robustly induced during BAC differentiation. Importantly, Prdm9 inactivation by shRNA knockdown or a selective inhibitor attenuates glutamine-triggered adipogenic and thermogenic induction. Furthermore, Prdm9 gene transcription is regulated by glutamine through the recruitment of C/EBPb to its enhancer region. This work reveals glutamine as a novel activator of thermogenic adipocyte differentiation and uncovers an unexpected role of C/EBPb-Prdm9-mediated H3K4me3 and transcriptional reprogramming in adipocyte differentiation and thermogenesis.

Ootheca mantidis mitigates renal fibrosis in mice by the suppression of apoptosis via increasing the gut microbe Akkermansia muciniphila and modulating glutamine metabolism.

Renal interstitial fibrosis (RIF), a progressive process affecting the kidneys in chronic kidney disease (CKD), currently lacks an effective therapeutic intervention. Traditional Chinese medicine (TCM) has shown promise in reducing RIF and slowing CKD progression. In this study, we demonstrated the dose-dependent attenuation of RIF by Ootheca mantidis (SPX), a commonly prescribed TCM for CKD, in a mouse model of unilateral ureteral obstruction (UUO). RNA-sequencing analysis suggested that SPX treatment prominently downregulated apoptosis and inflammation-associated pathways, thereby inhibiting the fibrogenic signaling in the kidney. We further found that transplantation of fecal microbiota from SPX-treated mice conferred protection against renal injury and fibrosis through suppressing apoptosis in UUO mice, indicating that SPX ameliorated RIF via remodeling the gut microbiota and reducing apoptosis in the kidneys. Further functional exploration of the gut microbiota combined with fecal metabolomics revealed increased levels of some probiotics, including Akkermansia muciniphila (A. muciniphila), and modulations in glutamine-related amino acid metabolism in UUO mice treated with SPX. Subsequent colonization of A. muciniphila and supplementation with glutamine effectively mitigated cell apoptosis and RIF in UUO mice. Collectively, these findings unveil a functionally A. muciniphila- and glutamine-involved gut-renal axis that contributes to the action of SPX, and provide important clue for the therapeutic potential of SPX, A. muciniphila, and glutamine in combatting RIF.