Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

INTRODUCTION: Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. OBJECTIVES: To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. METHODS: A dual extraction method involving 80% methanol followed by MTBE (methyl tert-butyl ether) extraction enables the analysis of free fatty acids, polar metabolites, and lipids. Extracts from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days were analyzed using HILIC chromatography coupled to Q Exactive Plus mass spectrometer or reversed-phase liquid chromatography coupled to MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. RESULTS: Lipidomics analysis of 6 mouse tissues and plasma allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that (1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; (2) the impact on lysophosphatidylcholine (lysoPC) and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations; (3) increase of intestinal tricarboxylic acid (TCA) cycle intermediates after metformin treatment. CONCLUSION: The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Targeting glutamine metabolism improves sarcoma response to radiation therapy in vivo.

Diverse tumor metabolic phenotypes are influenced by the environment and genetic lesions. Whether these phenotypes extend to rhabdomyosarcoma (RMS) and how they might be leveraged to design new therapeutic approaches remains an open question. Thus, we utilized a Pax7Cre-ER-T2/+; NrasLSL-G12D/+; p53fl/fl (P7NP) murine model of sarcoma with mutations that most frequently occur in human embryonal RMS. To study metabolism, we infuse 13C-labeled glucose or glutamine into mice with sarcomas and show that sarcomas consume more glucose and glutamine than healthy muscle tissue. However, we reveal a marked shift from glucose consumption to glutamine metabolism after radiation therapy (RT). In addition, we show that inhibiting glutamine, either through genetic deletion of glutaminase (Gls1) or through pharmacological inhibition of glutaminase, leads to significant radiosensitization in vivo. This causes a significant increase in overall survival for mice with Gls1-deficient compared to Gls1-proficient sarcomas. Finally, Gls1-deficient sarcomas post-RT elevate levels of proteins involved in natural killer cell and interferon alpha/gamma responses, suggesting a possible role of innate immunity in the radiosensitization of Gls1-deficient sarcomas. Thus, our results indicate that glutamine contributes to radiation response in a mouse model of RMS.

Inoculation fermentation with Lactobacillus fermentum L28 and Staphylococcus epidermidis S24 for improving the protein degradation of air-dried goose.

The inoculation fermentation technology was applied to the processing of dried cured goose to investigate the protein degradation. Lactobacillus fermentum (L), Staphylococcus epidermidis (S) and mixed strains (L + S) were individually inoculated into the whole goose before drying. We studied the degradation of protein in the air-dried period of goose. The results showed that compared with natural fermentation, inoculation fermentation significantly increased the content of non-protein nitrogen (14.85 mg/g NPN), proteolysis index (8.98% PI), myofibril fragmentation index (89.35 MFI) and total amount of free amino acids (1332.6 mg/g FAA) of dried cured goose. Electrophoresis revealed that the inoculation fermentation accelerated the degradation of macromolecular proteins and the accumulation of small molecular proteins. The degree of protein degradation in four groups of goose was in an order of L + S group > S group > L group > CK group. It suggested that inoculation fermentation could promote the degradation of myofibrillar proteins.

Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation.

Activating the macrophage NLRP3 inflammasome can promote excessive inflammation with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and interleukin-1ß (IL-1ß) secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves crista ultrastructure, and attenuates mitochondrial reactive oxygen species (ROS) production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. Our study suggests a non-canonical role of mitochondrial PDHK in promoting mitochondrial stress and supporting NLRP3 inflammasome activation during acute inflammation.

Data-dependent and -independent acquisition lipidomics analysis reveals the tissue-dependent effect of metformin on lipid metabolism.

Introduction: Despite the well-recognized health benefits, the mechanisms and site of action of metformin remains elusive. Metformin-induced global lipidomic changes in plasma of animal models and human subjects have been reported. However, there is a lack of systemic evaluation of metformin-induced lipidomic changes in different tissues. Metformin uptake requires active transporters such as organic cation transporters (OCTs), and hence, it is anticipated that metformin actions are tissue-dependent. In this study, we aim to characterize metformin effects in non-diabetic male mice with a special focus on lipidomics analysis. The findings from this study will help us to better understand the cell-autonomous (direct actions in target cells) or non-cell-autonomous (indirect actions in target cells) mechanisms of metformin and provide insights into the development of more potent yet safe drugs targeting a particular organ instead of systemic metabolism for metabolic regulations without major side effects. Objectives: To characterize metformin-induced lipidomic alterations in different tissues of non-diabetic male mice and further identify lipids affected by metformin through cell-autonomous or systemic mechanisms based on the correlation between lipid alterations in tissues and the corresponding in-tissue metformin concentrations. Methods: Lipids were extracted from tissues and plasma of male mice treated with or without metformin in drinking water for 12 days and analyzed using MS/MS scan workflow (hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer using biologically relevant lipids-containing inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow followed by data-dependent acquisition (DDA), to maximum the coverage of lipids and minimize the negative effect of stochasticity of precursor selection on experimental consistency and reproducibility. Results: Lipidomics analysis of 6 mouse tissues and plasma using MS/MS combining BRI-DIA and DDA allowed a systemic evaluation of lipidomic changes induced by metformin in different tissues. We observed that 1) the degrees of lipidomic changes induced by metformin treatment overly correlated with tissue concentrations of metformin; 2) the impact on lysophosphorylcholine and cardiolipins was positively correlated with tissue concentrations of metformin, while neutral lipids such as triglycerides did not correlate with the corresponding tissue metformin concentrations. Conclusion: The data collected in this study from non-diabetic mice with 12-day metformin treatment suggest that the overall metabolic effect of metformin is positively correlated with tissue concentrations and the effect on individual lipid subclass is via both cell-autonomous mechanisms (cardiolipins and lysoPC) and non-cell-autonomous mechanisms (triglycerides).

Immunoediting instructs tumor metabolic reprogramming to support immune evasion.

Immunoediting sculpts immunogenicity and thwarts host anti-tumor responses in tumor cells during tumorigenesis; however, it remains unknown whether metabolic programming of tumor cells can be guided by immunosurveillance. Here, we report that T cell-mediated immunosurveillance in early-stage tumorigenesis instructs c-Myc upregulation and metabolic reprogramming in tumor cells. This previously unexplored tumor-immune interaction is controlled by non-canonical interferon gamma (IFNγ)-STAT3 signaling and supports tumor immune evasion. Our findings uncover that immunoediting instructs deregulated bioenergetic programs in tumor cells to empower them to disarm the T cell-mediated immunosurveillance by imposing metabolic tug-of-war between tumor and infiltrating T cells and forming the suppressive tumor microenvironment.

13C tracer analysis suggests extensive recycling of endogenous CO2 in vivo.

BACKGROUND: 13C tracer analysis is increasingly used to monitor cellular metabolism in vivo and in intact cells, but data interpretation is still the key element to unveil the complexity of metabolic activities. The distinct 13C labeling patterns (e.g., M + 1 species in vivo but not in vitro) of metabolites from [U-13C]-glucose or [U-13C]-glutamine tracing in vivo and in vitro have been previously reported by multiple groups. However, the reason for the difference in the M + 1 species between in vivo and in vitro experiments remains poorly understood. METHODS: We have performed [U-13C]-glucose and [U-13C]-glutamine tracing in sarcoma-bearing mice (in vivo) and in cancer cell lines (in vitro). 13C enrichment of metabolites in cultured cells and tissues was determined by LC coupled with high-resolution mass spectrometry (LC-HRMS). All p-values are obtained from the Student's t-test two-tailed using GraphPad Prism 8 unless otherwise noted. RESULTS: We observed distinct enrichment patterns of tricarboxylic acid cycle intermediates in vivo and in vitro. As expected, citrate M + 2 or M + 4 was the dominant mass isotopologue in vitro. However, citrate M + 1 was unexpectedly the dominant isotopologue in mice receiving [U-13C]-glucose or [U-13C]-glutamine infusion, but not in cultured cells. Our results are consistent with a model where the difference in M + 1 species is due to the different sources of CO2 in vivo and in vitro, which was largely overlooked in the past. In addition, a time course study shows the generation of high abundance citrate M + 1 in plasma of mice as early as few minutes after [U-13C]-glucose infusion. CONCLUSIONS: Altogether, our results show that recycling of endogenous CO2 is substantial in vivo. The production and recycling of 13CO2 from the decarboxylation of [U-13C]-glucose or [U-13C]-glutamine is negligible in vitro partially due to dilution by the exogenous HCO3-/CO2 source, but in vivo incorporation of endogenous 13CO2 into M + 1 metabolites is substantial and should be considered. These findings provide a new paradigm to understand carbon atom transformations in vivo and should be taken into account when developing mathematical models to better reflect carbon flux.

Prioritize biologically relevant ions for data-independent acquisition (BRI-DIA) in LC-MS/MS-based lipidomics analysis.

INTRODUCTION: Data-dependent acquisition (DDA) is the most commonly used MS/MS scan method for lipidomics analysis on orbitrap-based instrument. However, MS instrument associated software decide the top N precursors for fragmentation, resulting in stochasticity of precursor selection and compromised consistency and reproducibility. We introduce a novel workflow using biologically relevant lipids to construct inclusion list for data-independent acquisition (DIA), named as BRI-DIA workflow. OBJECTIVES: To ensure consistent coverage of biologically relevant lipids in LC-MS/MS-based lipidomics analysis. METHODS: Biologically relevant ion list was constructed based on LIPID MAPS and lipidome atlas in MS-DIAL 4. Lipids were extracted from mouse tissues and used to assess different MS/MS scan workflow (DDA, BRI-DIA, and hybrid mode) on LC-Orbitrap Exploris 480 mass spectrometer. RESULTS: DDA resulted in more MS/MS events, but the total number of unique lipids identified by three methods (DDA, BRI-DIA, and hybrid MS/MS scan mode) is comparable (580 unique lipids across 44 lipid subclasses in mouse liver). Major cardiolipin molecular species were identified by data generated using BRI-DIA and hybrid methods and allowed calculation of cardiolipin compositions, while identification of the most abundant cardiolipin CL72:8 was missing in data generated using DDA method, leading to wrong calculation of cardiolipin composition. CONCLUSION: The method of using inclusion list comprised of biologically relevant lipids in DIA MS/MS scan is as efficient as traditional DDA method in profiling lipids, but offers better consistency of lipid identification, compared to DDA method. This study was performed using Orbitrap Exploris 480, and we will further evaluate this workflow on other platforms, and if verified by future work, this biologically relevant ion fragmentation workflow could be routinely used in many studies to improve MS/MS identification capacities.

Tigliane Diterpenoids with Larvicidal, Antifungal, and α-Glucosidase Inhibitory Activities from Croton damayeshu.

Thirty-five tigliane diterpenoids and two ent-kaurane diterpenoids were isolated from the leaves of Croton damayeshu, and, among them, compounds 1-10 were characterized as new tigliane diterpenoids. The structures of compounds 1-10 were determined by analysis of their HRESIMS, NMR, and ECD data and by chemical methods. The isolates were assayed for their larvicidal, antifungal, and α-glucosidase inhibitory activities, and compounds 8-10 were found to possess larvicidal activities against Plutella xylostella with LC50 values of 0.19, 0.16, and 0.26 µM, respectively, comparable to the LC50 of 0.14 µM for the positive control, flubendiamide.

Four new triterpene glucosides from Salacia cochinchinensis Lour.

Four new triterpene glucosides (1-4) were isolated from the 90% ethanol extract of Salacia cochinchinensis, together with five known compounds (5-9). The structures of the new compounds were elucidated by comprehensive spectroscopic analysis including HRESIMS, IR, 1 D and 2 D NMR analysis. All isolates were assayed for their α-glucosidase inhibitory activity. Compound 9 showed remarkable α-glucosidase inhibitory activity with an IC50 value of 0.31 µM, and the triterpene glycosides (1-5) exhibited moderate α-glucosidase inhibitory activity.

Reinforcing one-carbon metabolism via folic acid/Folr1 promotes ß-cell differentiation.

Diabetes can be caused by an insufficiency in ß-cell mass. Here, we performed a genetic screen in a zebrafish model of ß-cell loss to identify pathways promoting ß-cell regeneration. We found that both folate receptor 1 (folr1) overexpression and treatment with folinic acid, stimulated ß-cell differentiation in zebrafish. Treatment with folinic acid also stimulated ß-cell differentiation in cultures of neonatal pig islets, showing that the effect could be translated to a mammalian system. In both zebrafish and neonatal pig islets, the increased ß-cell differentiation originated from ductal cells. Mechanistically, comparative metabolomic analysis of zebrafish with/without ß-cell ablation and with/without folinic acid treatment indicated ß-cell regeneration could be attributed to changes in the pyrimidine, carnitine, and serine pathways. Overall, our results suggest evolutionarily conserved and previously unknown roles for folic acid and one-carbon metabolism in the generation of ß-cells.

Molecularly Imprinted Micelles for Fluorescent Sensing of Nonsteroidal Anti-Inflammatory Drugs (NSAIDs).

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used over-the-counter drugs and their uncontrolled disposal is a significant environmental concern. Although their fluorescent sensing is a desirable method of detection for its sensitivity and simplicity, the structural similarity of the drugs makes the design of selective sensors highly challenging. A thiourea-based fluorescent functional monomer was identified in this work to enable highly efficient synthesis of molecularly imprinted nanoparticle (MINP) sensors for NSAIDs such as Indomethacin or Tolmetin. Micromolar binding affinities were obtained in aqueous solution, with binding selectivities comparable to those reported for polyclonal antibodies. The detection limit was ~50 ng/mL in aqueous solution, and common carboxylic acids such as acetic acid, benzoic acid, and citric acid showed negligible interference.

Disruption of microRNA-214 during general anaesthesia prevents brain injury and maintains mitochondrial fusion by promoting Mfn2 interaction with Pkm2.

Duration of surgical general anaesthesia is associated with severe brain injury and neurological deficits. The specific mechanisms underlying post-general anaesthesia brain injury, however, still remain to be elucidated. Herein, we explore the role of microRNA-214 (miR-214) in the occurrence of brain injury after general anaesthesia and its underlying mechanism. Hippocampal tissues and neurons were isolated from rats exposed to 2% sevoflurane. TUNEL stains reflect hippocampal neuron apoptosis. Cultured hippocampal neurons stained with JC-1 and MitoTracker dyes were imaged by fluorescence microscope to visualize changes of mitochondrial membrane potential and mitochondrial fusion. Mitochondrial function was evaluated. Mitofusin 2 (Mfn2) binding to miR-214 or pyruvate kinase M2 (Pkm2) was confirmed by co-immunoprecipitation, immunofluorescence, dual luciferase reporter gene and RNA immunoprecipitation assays. After exposure to 2% sevoflurane, up-regulated miR-214 expression and impaired interaction between Mfn2 and Pkm2 were found in rat hippocampal tissues. Rats exposed to 2% sevoflurane also experienced neuronal injury, mitochondrial defects and deficits in the brain-derived neurotrophic factor (Bdnf) signalling. miR-214 was shown to target Mfn2 by impairing its binding with Pkm2. Inhibiting miR-214 expression using its specific inhibitor improved mitochondrial membrane potential, enhanced mitochondrial fusion, maintained mitochondrial function, restored interaction between Mfn2 and Pkm2, and activated the Bdnf signalling in cultured hippocampal neurons. Adenovirus infection of miR-214 inhibitor reduced neuron apoptosis and maintained mitochondrial function in the hippocampus of rats exposed to 2% sevoflurane. Taken together, the study demonstrates inhibition of miR-214 is cerebral protective against brain injury following general anaesthesia.

Synthetic lectins for selective binding of glycoproteins in water.

Although synthetic mimics of lectins can be extremely useful in biological and biomedical research, molecular recognition of carbohydrates has been hampered by their strong solvation in water and subtle structural differences among analogues. Molecularly imprinted nanoparticle receptors were prepared with glycans directly cleaved from glycoproteins. Functionalized with boroxole groups in the binding sites, these water-soluble synthetic lectins bound the parent glycoproteins selectively in water with an association constant of Ka = 104-105 M-1. The strong binding enabled the receptors to protect the targeted glycans from enzymatic cleavage. When clicked onto magnetic nanoparticles, the receptors enabled facile isolation of glycoproteins from a mixture.

Selective Binding of Dopamine and Epinephrine in Water by Molecularly Imprinted Fluorescent Receptors.

Catecholamines play important roles in biology but their structural similarity makes it challenging to construct synthetic receptors with selective binding. A combination of covalent and noncovalent binding groups in the hydrophobic core of water-soluble nanoparticles enabled them to recognize dopamine and epinephrine with an association constant (Ka ) of 3-4×104  M-1 in water, an order of magnitude higher than those of previously reported synthetic hosts. In addition, minute structural changes among analogues were detected including the addition or removal of a single hydroxyl or methyl group.

Zwitterionic Molecularly Imprinted Cross-Linked Micelles for Alkaloid Recognition in Water.

Molecular imprinting within surface/core doubly cross-linked micelles afforded water-soluble nanoparticle receptors for their template molecules. Extremely strong imprinting effects were consistently observed, with the imprinting factor >100:1 in comparison to nonimprinted nanoparticles prepared without the templates. The ionic nature of the cross-linkable surfactant strongly impacted the imprinting and binding process. Imprinted receptors prepared with a zwitterionic cross-linkable surfactant (4) outperformed a similar cationic one (1) when the template was zwitterionic or cationic and preferred their templates over structural analogues regardless of their ionic characteristics. Electrostatic interactions, however, dominated the receptors made with the cationic surfactant. The same micellar imprinting applied to simple as well as complex alkaloids. Imprinted receptors from 4 were also shown to categorize their alkaloid guests according to their structural similarity.

Selective Binding of Folic Acid and Derivatives by Imprinted Nanoparticle Receptors in Water.

Folate receptors are overexpressed on cancer cells and frequently used for targeted delivery. Creation of synthetic receptors to bind folic acid and its analogues in water, however, is challenging because of its complex hydrogen-bonding patterns and competition for hydrogen bonds from the solvent. Micellar imprinting within cross-linkable surfactants circumvented these problems because the nonpolar micellar environment strengthened the hydrogen bonds between the amide group in the surfactant and the template molecule. Incorporation of polymerizable thiouronium functional monomers further enhanced the binding through hydrogen-bond-reinforced ion pairs with the glutamate moiety of the template. The resulting imprinted micelles were able to bind folate and their analogues with submicromolar affinity and distinguish small changes in the hydrogen-bonding patterns as well as the number/position of carboxylic acids. The binding constant obtained was 2-3 orders of magnitude higher than those reported for small-molecule synthetic receptors. Our binding study also revealed interesting details in the binding. For example, the relative contributions of different segments of the molecule to the binding followed the order of carboxylates > pyrimidine ring > pyrazine ring.

Peroxides as "switches" of dialkyl H-phosphonate: two mild and metal-free methods for preparation of 2-acylbenzothiazoles and dialkyl benzothiazol-2-ylphosphonates.

Two mild and metal-free methods for the preparation of two kinds of important benzothiazole derivatives, 2-acylbenzothiazoles and dialkyl benzothiazol-2-ylphosphonates, respectively, were developed. The dialkyl H-phosphonate (RO)2P(O)H exists in equilibrium with its tautomer dialkyl phosphite (RO)2POH. TBHP triggered α-carbon-centered phosphite radical formation, whereas DTBP triggered phosphorus-centered phosphonate radical formation. The two types of radicals led respectively to two different reaction processes, the direct C2-acylation of benzothiazoles and C2-phosphonation of benzothiazoles.

Silver catalyzed decarboxylative direct C2-alkylation of benzothiazoles with carboxylic acids.

A novel and efficient silver catalyzed decarboxylative direct C2-alkylation of benzothiazoles with carboxylic acids for the synthesis of 2-alkyl benzothiazoles was developed.