Mechanistic insight into allosteric activation of human pyruvate carboxylase by acetyl-CoA.

Pyruvate carboxylase (PC) catalyzes the two-step carboxylation of pyruvate to produce oxaloacetate, playing a key role in the maintenance of metabolic homeostasis in cells. Given its involvement in multiple diseases, PC has been regarded as a potential therapeutic target for obesity, diabetes, and cancer. Albeit acetyl-CoA has been recognized as the allosteric regulator of PC for over 60 years, the underlying mechanism of how acetyl-CoA induces PC activation remains enigmatic. Herein, by using time-resolved cryo-electron microscopy, we have captured the snapshots of PC transitional states during its catalytic cycle. These structures and the biochemical studies reveal that acetyl-CoA stabilizes PC in a catalytically competent conformation, which triggers a cascade of events, including ATP hydrolysis and the long-distance communication between the two reactive centers. These findings provide an integrated picture for PC catalysis and unveil the unique allosteric mechanism of acetyl-CoA in an essential biochemical reaction in all kingdoms of life.

Standing genetic variation fuels rapid evolution of herbicide resistance in blackgrass.

Repeated herbicide applications in agricultural fields exert strong selection on weeds such as blackgrass (Alopecurus myosuroides), which is a major threat for temperate climate cereal crops. This inadvertent selection pressure provides an opportunity for investigating the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much target-site resistance (TSR) relies on de novo mutations vs. standing variation is important for developing strategies to manage herbicide resistance. We first generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Next, through empirical data in the form of highly accurate long-read amplicons of alleles encoding acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) variants, we showed that most populations with resistance due to TSR mutations-23 out of 27 and six out of nine populations for ACCase and ALS, respectively-contained at least two TSR haplotypes, indicating that soft sweeps are the norm. Finally, through forward-in-time simulations, we inferred that TSR is likely to mainly result from standing genetic variation, with only a minor role for de novo mutations.

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast.

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.

CARS senses cysteine deprivation to activate AMPK for cell survival.

Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is an important cellular metabolite-sensing enzyme that can directly sense changes not only in ATP but also in metabolites associated with carbohydrates and fatty acids. However, less is known about whether and how AMPK senses variations in cellular amino acids. Here, we show that cysteine deficiency significantly triggers calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-mediated activation of AMPK. In addition, we found that CaMKK2 directly associates with cysteinyl-tRNA synthetase (CARS), which then binds to AMPKγ2 under cysteine deficiency to activate AMPK. Interestingly, we discovered that cysteine inhibits the binding of CARS to AMPKγ2, and thus, under cysteine deficiency conditions wherein the inhibitory effect of cysteine is abrogated, CARS mediates the binding of AMPK to CaMKK2, resulting in the phosphorylation and activation of AMPK by CaMKK2. Importantly, we demonstrate that blocking AMPK activation leads to cell death under cysteine-deficient conditions. In summary, our study is the first to show that CARS senses the absence of cysteine and activates AMPK through the cysteine-CARS-CaMKK2-AMPKγ2 axis, a novel adaptation strategy for cell survival under nutrient deprivation conditions.

ACC1-mediated fatty acid biosynthesis intrinsically controls thymic iNKT cell development.

To meet the energetic requirements associated with activation, proliferation, and survival, T cells switch their metabolic signatures from energetically quiescent to activated. However, little is known about the role of metabolic pathway controlling the development of invariant natural killer T (iNKT) cells. In the present study, we found that acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme for the fatty acid biosynthesis pathway, plays an essential role in the development of iNKT cells in the thymus. Mice lacking T-cell specific ACC1 showed a reduced number of iNKT cells with an increased proportion of iNKT cells at immature stages 0 and 1. Furthermore, mixed bone marrow (BM) chimera experiments revealed that T-cell intrinsic ACC1 expression was selectively important for the development of thymic iNKT cells, especially for the differentiation of the NKT1 cell subset. Our single-cell RNA-sequencing (scRNA-seq) data and functional analysis demonstrated that ACC1 is responsible for survival of developing iNKT cells. Thus, these findings highlighted a novel role of ACC1 in controlling thymic iNKT cell development mediated by the control of cell survival.

Activation of hepatic acetyl-CoA carboxylase by S-nitrosylation in response to diet.

Nitric oxide (NO), produced primarily by nitric oxide synthase enzymes, is known to influence energy metabolism by stimulating fat uptake and oxidation. The effects of NO on de novo lipogenesis (DNL), however, are less clear. Here we demonstrate that hepatic expression of endothelial nitric oxide synthase is reduced following prolonged administration of a hypercaloric high-fat diet. This results in marked reduction in the amount of S-nitrosylation of liver proteins including notably acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in DNL. We further show that ACC S-nitrosylation markedly increases enzymatic activity. Diminished endothelial nitric oxide synthase expression and ACC S-nitrosylation may thus represent a physiological adaptation to caloric excess by constraining lipogenesis. Our findings demonstrate that S-nitrosylation of liver proteins is subject to dietary control and suggest that DNL is coupled to dietary and metabolic conditions through ACC S-nitrosylation.

Acetyl-CoA carboxylase 1 depletion suppresses de novo fatty acid synthesis and mitochondrial ß-oxidation in castration-resistant prostate cancer cells.

Cancer cells, including those of prostate cancer (PCa), often hijack intrinsic cell signaling to reprogram their metabolism. Part of this reprogramming includes the activation of de novo synthesis of fatty acids that not only serve as building blocks for membrane synthesis but also as energy sources for cell proliferation. However, how de novo fatty acid synthesis contributes to PCa progression is still poorly understood. Herein, by mining public datasets, we discovered that the expression of acetyl-CoA carboxylase alpha (ACACA), which encodes acetyl-CoA carboxylase 1 (ACC1), was highly expressed in human PCa. In addition, patients with high ACACA expression had a short disease-free survival time. We also reported that depletion of ACACA reduced de novo fatty acid synthesis and PI3K/AKT signaling in the human castration-resistant PCa (CRPC) cell lines DU145 and PC3. Furthermore, depletion of ACACA downregulates mitochondrial beta-oxidation, resulting in mitochondrial dysfunction, a reduction in ATP production, an imbalanced NADP+/NADPhydrogen(H) ratio, increased reactive oxygen species, and therefore apoptosis. Reduced exogenous fatty acids by depleting lipid or lowering serum supplementation exacerbated both shRNA depletion and pharmacological inhibition of ACACA-induced apoptosis in vitro. Collectively, our results suggest that inhibition of ectopic ACACA, together with suppression of exogenous fatty acid uptake, can be a novel strategy for treating currently incurable CRPC.

Repurposing a human acetyl-CoA carboxylase inhibitor firsocostat to treat fungal candidiasis alone and in combination.

Opportunistic fungal infections, particularly caused by Candida albicans, remain a common cause of high morbidity and mortality in immunocompromised patients. The escalating prevalence of antifungal drug resistance necessitates the immediate exploration of alternative treatment strategies to combat these life-threatening fungal diseases. In this study, we investigated the antifungal efficacy of firsocostat, a human acetyl-CoA carboxylase (ACC) inhibitor, against C. albicans. Firsocostat alone displayed moderate antifungal activity, while combining it with voriconazole, itraconazole, or amphotericin B exhibited synergistic effects across almost all drug-sensitive and drug-resistant C. albicans strains tested. These observed synergies were further validated in two mouse models of oropharyngeal and systemic candidiasis, where the combination therapies demonstrated superior fungicidal effects compared to monotherapy. Moreover, firsocostat was shown to directly bind to C. albicans ACC and inhibit its enzymatic activity. Sequencing spontaneous firsocostat-resistant mutants revealed mutations mapping to C. albicans ACC, confirming that firsocostat has retained its target in C. albicans. Overall, our findings suggest that repurposing firsocostat, either alone or in combination with other antifungal agents, holds promising potential in the development of antifungal drugs and the treatment of candidiasis.

ACACA reduces lipid accumulation through dual regulation of lipid metabolism and mitochondrial function via AMPK- PPARα- CPT1A axis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a multifaceted metabolic disorder, whose global prevalence is rapidly increasing. Acetyl CoA carboxylases 1 (ACACA) is the key enzyme that controls the rate of fatty acid synthesis. Hence, it is crucial to investigate the function of ACACA in regulating lipid metabolism during the progress of NAFLD. METHODS: Firstly, a fatty liver mouse model was established by high-fat diet at 2nd, 12th, and 20th week, respectively. Then, transcriptome analysis was performed on liver samples to investigate the underlying mechanisms and identify the target gene of the occurrence and development of NAFLD. Afterwards, lipid accumulation cell model was induced by palmitic acid and oleic acid (PA ∶ OA molar ratio = 1∶2). Next, we silenced the target gene ACACA using small interfering RNAs (siRNAs) or the CMS-121 inhibitor. Subsequently, experiments were performed comprehensively the effects of inhibiting ACACA on mitochondrial function and lipid metabolism, as well as on AMPK- PPARα- CPT1A pathway. RESULTS: This data indicated that the pathways significantly affected by high-fat diet include lipid metabolism and mitochondrial function. Then, we focus on the target gene ACACA. In addition, the in vitro results suggested that inhibiting of ACACA in vitro reduces intracellular lipid accumulation, specifically the content of TG and TC. Furthermore, ACACA ameliorated mitochondrial dysfunction and alleviate oxidative stress, including MMP complete, ATP and ROS production, as well as the expression of mitochondria respiratory chain complex (MRC) and AMPK proteins. Meanwhile, ACACA inhibition enhances lipid metabolism through activation of PPARα/CPT1A, leading to a decrease in intracellular lipid accumulation. CONCLUSION: Targeting ACACA can reduce lipid accumulation by mediating the AMPK- PPARα- CPT1A pathway, which regulates lipid metabolism and alleviates mitochondrial dysfunction.

Low phosphorus increases hepatic lipid deposition, oxidative stress and inflammatory response via Acetyl-CoA carboxylase-dependent manner in zebrafish liver cells.

Acetyl-CoA carboxylase (ACC) plays a regulatory role in both fatty acid synthesis and oxidation, controlling the process of lipid deposition in the liver. Given that existing studies have shown a close relationship between low phosphorus (P) and hepatic lipid deposition, this study was conducted to investigate whether ACC plays a crucial role in this relationship. Zebrafish liver cell line (ZFL) was incubated under low P medium (LP, P concentration: 0.77 mg/L) or adequate P medium (AP, P concentration: 35 mg/L) for 240 h. The results showed that, compared with AP-treated cells, LP-treated cells displayed elevated lipid accumulation, and reduced fatty acid ß-oxidation, ATP content, and mitochondrial mass. Furthermore, transcriptomics analysis revealed that LP-treated cells significantly increased lipid synthesis (Acetyl-CoA carboxylases (acc), Stearyl coenzyme A dehydrogenase (scd)) but decreased fatty acid ß-oxidation (Carnitine palmitoyltransferase I (cptI)) and (AMP-activated protein kinase (ampk)) mRNA levels compared to AP-treated cells. The phosphorylation of AMPK and ACC, and the protein expression of CPTI were significantly decreased in LP-treated cells compared with those in AP-treated cells. After 240 h of LP treatment, PF-05175157 (an ACC inhibitor) was supplemented in the LP treatment for an additional 12 h. PF-05175157-treated cells showed higher phosphorylation of ACC, higher protein expression of CPTI, and lower protein expression of FASN, lower TG content, enhanced fatty acid ß-oxidation, increased ATP content, and mitochondrial mass compared with LP-treated cells. PF-05175157 also relieved the LP-induced oxidative stress and inflammatory response. Overall, these findings suggest that ACC is a promising target for treating LP-induced elevation of lipid deposition in ZFL, and can alleviate oxidative stress and inflammatory response.

Molecular cloning and gene expression of acc2 from grass carp (Ctenopharyngodon idella) and the regulation of glucose metabolism by ACCs inhibitor.

BACKGROUND: Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to malonyl-CoA. Malonyl-CoA, which plays a key role in regulating glucose and lipid metabolism, is not only a substrate for fatty acid synthesis but also an inhibitor of the oxidation pathway. ACC exists as two isoenzymes that are encoded by two different genes. ACC1 in grass carp (Ctenopharyngodon idellus) has been cloned and sequenced. However, studies on the cloning, tissue distribution, and function of ACC2 in grass carp were still rare. METHODS AND RESULTS: The full-length cDNA of acc2 was 8537 bp with a 7146 bp open reading frame encoding 2381 amino acids. ACC2 had a calculated molecular weight of 268.209 kDa and an isoelectric point of 5.85. ACC2 of the grass carp shared the closest relationship with that of the common carp (Sinocyclocheilus grahami). The expressions of acc1 and acc2 mRNA were detected in all examined tissues.  The expression level of acc1 was high in the brain and fat but absent in the midgut and hindgut. The expression level of acc2 in the kidney was significantly higher than in other tissues, followed by the heart, brain, muscle, and spleen. ACCs inhibitor significantly reduced the levels of glucose, malonyl-CoA, and triglyceride in hepatocytes. CONCLUSIONS: This study showed that the function of ACC2 was evolutionarily conserved from fish to mammals. ACCs inhibitor inhibited the biological activity of ACCs, and reduced fat accumulation in grass carp.

Structural basis for regulation of human acetyl-CoA carboxylase.

Acetyl-CoA carboxylase catalyses the ATP-dependent carboxylation of acetyl-CoA, a rate-limiting step in fatty acid biosynthesis1,2. Eukaryotic acetyl-CoA carboxylases are large, homodimeric multienzymes. Human acetyl-CoA carboxylase occurs in two isoforms: the metabolic, cytosolic ACC1, and ACC2, which is anchored to the outer mitochondrial membrane and controls fatty acid ß-oxidation1,3. ACC1 is regulated by a complex interplay of phosphorylation, binding of allosteric regulators and protein-protein interactions, which is further linked to filament formation1,4-8. These filaments were discovered in vitro and in vivo 50 years ago7,9,10, but the structural basis of ACC1 polymerization and regulation remains unknown. Here, we identify distinct activated and inhibited ACC1 filament forms. We obtained cryo-electron microscopy structures of an activated filament that is allosterically induced by citrate (ACC-citrate), and an inactivated filament form that results from binding of the BRCT domains of the breast cancer type 1 susceptibility protein (BRCA1). While non-polymeric ACC1 is highly dynamic, filament formation locks ACC1 into different catalytically competent or incompetent conformational states. This unique mechanism of enzyme regulation via large-scale conformational changes observed in ACC1 has potential uses in engineering of switchable biosynthetic systems. Dissecting the regulation of acetyl-CoA carboxylase opens new paths towards counteracting upregulation of fatty acid biosynthesis in disease.

Improving the substrate binding of acetyl-CoA carboxylase (AccB) from Streptomyces antibioticus through computational enzyme engineering.

Malonyl-CoA serves as the main building block for the biosynthesis of many important polyketides, as well as fatty acid-derived compounds, such as biofuel. Escherichia coli, Corynebacterium gultamicum, and Saccharomyces cerevisiae have recently been engineered for the biosynthesis of such compounds. However, the developed processes and strains often have insufficient productivity. In the current study, we used enzyme-engineering approach to improve the binding of acetyl-CoA with ACC. We generated different mutations, and the impact was calculated, which reported that three mutations, that is, S343A, T347W, and S350W, significantly improve the substrate binding. Molecular docking investigation revealed an altered binding network compared to the wild type. In mutants, additional interactions stabilize the binding of the inner tail of acetyl-CoA. Using molecular simulation, the stability, compactness, hydrogen bonding, and protein motions were estimated, revealing different dynamic properties owned by the mutants only but not by the wild type. The findings were further validated by using the binding-free energy (BFE) method, which revealed these mutations as favorable substitutions. The total BFE was reported to be -52.66 ± 0.11 kcal/mol for the wild type, -55.87 ± 0.16 kcal/mol for the S343A mutant, -60.52 ± 0.25 kcal/mol for T347W mutant, and -59.64 ± 0.25 kcal/mol for the S350W mutant. This shows that the binding of the substrate is increased due to the induced mutations and strongly corroborates with the docking results. In sum, this study provides information regarding the essential hotspot residues for the substrate binding and can be used for application in industrial processes.

PHD3 Loss in Cancer Enables Metabolic Reliance on Fatty Acid Oxidation via Deactivation of ACC2.

While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.

Nicotinate-curcumin improves NASH by inhibiting the AKR1B10/ACCα-mediated triglyceride synthesis.

BACKGROUND: Nonalcoholic steatohepatitis (NASH) is a prevalent chronic liver condition. However, the potential therapeutic benefits and underlying mechanism of nicotinate-curcumin (NC) in the treatment of NASH remain uncertain. METHODS: A rat model of NASH induced by a high-fat and high-fructose diet was treated with nicotinate-curcumin (NC, 20, 40 mg·kg- 1), curcumin (Cur, 40 mg·kg- 1) and metformin (Met, 50 mg·kg- 1) for a duration of 4 weeks. The interaction between NASH, Cur and Aldo-Keto reductase family 1 member B10 (AKR1B10) was filter and analyzed using network pharmacology. The interaction of Cur, NC and AKR1B10 was analyzed using molecular docking techniques, and the binding energy of Cur and NC with AKR1B10 was compared. HepG2 cells were induced by Ox-LDL (25 µg·ml- 1, 24 h) in high glucose medium. NC (20µM, 40µM), Cur (40µM) Met (150µM) and epalrestat (Epa, 75µM) were administered individually. The activities of ALT, AST, ALP and the levels of LDL, HDL, TG, TC and FFA in serum were quantified using a chemiluminescence assay. Based on the changes in the above indicators, score according to NAS standards. The activities of Acetyl-CoA and Malonyl-CoA were measured using an ELISA assay. And the expression and cellular localization of AKR1B10 and Acetyl-CoA carboxylase (ACCα) in HepG2 cells were detected by Western blotting and immunofluorescence. RESULTS: The results of the animal experiments demonstrated that NASH rat model induced by a high-fat and high-fructose diet exhibited pronounced dysfunction in liver function and lipid metabolism. Additionally, there was a significant increase in serum levels of FFA and TG, as well as elevated expression of AKR1B10 and ACCα, and heightened activity of Acetyl-CoA and Malonyl-CoA in liver tissue. The administration of NC showed to enhance liver function in rats with NASH, leading to reductions in ALT, AST and ALP levels, and decrease in blood lipid and significant inhibition of FFA and TG synthesis in the liver. Network pharmacological analysis identified AKR1B10 and ACCα as potential targets for NASH treatment. Molecular docking studies revealed that both Cur and NC are capable of binding to AKR1B10, with NC exhibiting a stronger binding energy to AKR1B10. Western blot analysis demonstrated an upregulation in the expression of AKR1B10 and ACCα in the liver tissue of NASH rats, accompanied by elevated Acetyl-CoA and Malonyl-CoA activity, and increased levels of FFA and TG. The results of the HepG2 cell experiments induced by Ox-LDL suggest that NC significantly inhibited the expression and co-localization of AKR1B10 and ACCα, while also reduced levels of TC and LDL-C and increased level of HDL-C. These effects are accompanied by a decrease in the activities of ACCα and Malonyl-CoA, and levels of FFA and TG. Furthermore, the impact of NC appears to be more pronounced compared to Cur. CONCLUSION: NC could effectively treat NASH and improve liver function and lipid metabolism disorder. The mechanism of NC is related to the inhibition of AKR1B10/ACCα pathway and FFA/TG synthesis of liver.

Intestinal microbiota promoted NiONPs-induced liver fibrosis via effecting serum metabolism.

Nickel oxide nanoparticles (NiONPs) are toxic heavy metal compounds that induce liver fibrosis and metabolic disorders. Current research shows that the intestinal microbiota regulates liver metabolism through the gut-liver axis. However, it is unclear whether NiONPs affect the intestinal microbiota and the relationship between microbiota and liver metabolic disorders. Therefore, in this study, we established liver fibrosis model by administering 0.015, 0.06 and 0.24 mg/mL NiONPs through tracheal instillation twice a week for 9 weeks in rats, then we collected serum and fecal sample for whole metabolomics and metagenomic sequencing. As the result of sequencing, we screened out seven metabolites (beta-D-glucuronide, methylmalonic acid, linoleic acid, phosphotidylcholine, lysophosphatidylinositol, docosapentaenoic acid and progesterone) that related to functional alterations (p < 0.05), and obtained a decrease of probiotics abundances (p < 0.05) as well as a variation of the microbiota enzyme activity (p < 0.05), indicating that NiONPs inhibited the proliferation of probiotics. As the result of correlation analysis, we found a positive correlation between differential metabolites and probiotics, such as lysophosphatidylinositol was positively correlated with Desulfuribacillus, Jeotgallibacillus and Rummeliibacillus (p < 0.05). We also found that differential metabolites had correlations with differential proteins and enzymes of intestinal microbiota, such as glucarate dehydratase, dihydroorotate dehydrogenase and acetyl-CoA carboxylase (p < 0.05). Finally, we screened six metabolic pathways with both differential intestinal microbiota enzymes and metabolites were involved, such as pentose and glucuronate interconversions, and linoleic acid metabolism. In vitro experiments showed that NiONPs increased the transcriptional expression of Col1A1 in LX-2 cells, while reducing the mRNA expression of serine/threonine activators, acetyl coenzyme carboxylase, and lysophosphatidylinositol synthase, and short chain fatty acid sodium butyrate can alleviate these variation trends. The results proved that the intestinal microbiota enzyme systems were associated with serum metabolites, suggesting that the disturbance of intestinal microbiota and reduction of probiotics promoted the occurrence and development of NiONPs-induced liver fibrosis by affecting metabolic pathways.

The Cys-2088-Arg mutation in the ACCase gene and enhanced metabolism confer cyhalofop-butyl resistance in Chinese sprangletop (Leptochloa chinensis).

Acetyl-CoA carboxylase (ACCase)-inhibiting herbicides are among the most commonly used herbicides to control grassy weeds, especially Leptochloa chinensis, in rice fields across China. Herein, we collected a suspected resistant (R) population of L. chinensis (HFLJ16) from Lujiang county in Anhui Province. Whole plant dose response tests showed that, compared with the susceptible (S) population, the R population showed high resistance to cyhalofop-butyl (22-fold) and displayed cross-resistance to metamifop (9.7-fold), fenoxaprop-P-ethyl (18.7-fold), quizalofop-P-ethyl (7.6-fold), clodinafop-propargyl (12-fold) and clethodim (8.4-fold). We detected an amino acid substitution (Cys-2088-Arg) in the ACCase of resistant L. chinensis. However, ACCase gene expression levels were not significantly different (P > 0.05) between R plants and S plants, without or with cyhalofop-butyl treatment. Furthermore, pretreatment with piperonyl butoxide (PBO, a cytochrome P450 monooxygenase (CYP450) inhibitor) or 4-chloro-7-nitrobenzoxadiazole (NBD-Cl, a glutathione-S-transferase (GST) inhibitor), inhibited the resistance of the R population to cyhalofop-butyl significantly (by approximately 60% and 26%, respectively). Liquid chromatography tandem mass spectrometry analysis showed that R plants metabolized cyhalofop-butyl and cyhalofop acid (its metabolite) significantly faster than S plants. Three CYP450 genes, one GST gene, and two ABC transporter genes were induced by cyhalofop-butyl and were overexpressed in the R population. Overall, GST-associated detoxification, CYP450 enhancement, and target-site gene mutation are responsible for the resistance of L. chinensis to cyhalofop-butyl.

Fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon resistance status in Beckmannia syzigachne from wheat fields across Anhui Province, China.

Severe infestations of American sloughgrass (Beckmannia syzigachne (Steud.) Fernald) in wheat fields throughout Anhui Province, China, pose a significant threat to local agricultural production. This study aims to evaluate the susceptibility of 37 B. syzigachne populations collected from diverse wheat fields in Anhui Province to three commonly used herbicides: fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon. Single-dose testing revealed that out of the 37 populations, 31, 26, and 11 populations had either evolved or were evolving resistance to fenoxaprop-P-ethyl, mesosulfuron-ethyl, and isoproturon, respectively. Among them, 25 populations displayed concurrent resistance to both fenoxaprop-P-ethyl and mesosulfuron-ethyl, while eight exhibited resistance to all three tested herbicides. Whole-plant bioassays confirmed that approximately 84% of the fenoxaprop-P-ethyl-resistant populations manifested high-level resistance (resistance index (RI) ≥10); 62% of the mesosulfuron-ethyl-resistant populations and 82% of the isoproturon-resistant populations exhibited low- to moderate-level resistance (2 ≤ RI <10). Three distinct target-site mutations were identified in 27% of fenoxaprop-P-ethyl-resistant populations, with no known resistance mutations detected in the remaining herbicide-resistant populations. The inhibition of cytochrome P450s (P450s) and/or glutathione S-transferases (GSTs) substantially increased susceptibility in the majority of resistant populations lacking mutations at the herbicide target site. In conclusion, resistance to fenoxaprop-P-ethyl and mesosulfuron-ethyl was widespread in B. syzigachne within Anhui Province's wheat fields, while resistance to isoproturon was rapidly evolving due to its escalating usage. Target-site mutations were present in approximately one-third of fenoxaprop-P-ethyl-resistant populations, and alternative mechanisms involving P450s and/or GSTs could explain the resistance observed in most of the remaining populations.

Pin1 Exacerbates Non-Alcoholic Fatty Liver Disease by Enhancing Its Activity through Binding to ACC1.

Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by diffuse hepatocellular steatosis due to fatty deposits in hepatocytes, excluding alcohol and other known liver injury factors. However, there are no specific drugs for the clinical treatment of NAFLD. Therefore, research on the pathogenesis of NAFLD at the cellular and molecular levels is a promising approach to finding therapeutic targets and developing targeted drugs for NAFLD. Pin1 is highly expressed during adipogenesis and contributes to adipose differentiation, but its specific mechanism of action in NAFLD is unclear. In this study, we investigated the role of Pin1 in promoting the development of NAFLD and its potential mechanisms in vitro and in vivo. First, Pin1 was verified in the NAFLD model in vitro using MCD diet-fed mice by Western Blot, RT-qPCR and immunohistochemistry (IHC) assays. In the in vitro study, we used the oleic acid (OA) stimulation-induced lipid accumulation model and examined the lipid accumulation in each group of cells by oil red O staining as well as BODIPY staining. The results showed that knockdown of Pin1 inhibited lipid accumulation in hepatocytes in an in vitro lipid accumulation model and improved lipid indices and liver injury levels. Moreover, in vivo, WT and Pin1-KO mice were fed a methionine-choline deficient (MCD) diet for 4 weeks to induce the NAFLD model. The effects of Pin1 on lipid accumulation, hepatic fibrosis, and oxidative stress were evaluated by biochemical analysis, glucose and insulin tolerance tests, histological analysis, IHC, RT-qPCR and Western blot assays. The results indicate that Pin1 knockdown significantly alleviated hepatic steatosis, fibrosis and inflammation in MCD-induced NAFLD mice, improved glucose tolerance and alleviated insulin resistance in mice. Further studies showed that the AMPK/ACC1 signalling pathway might take part in the process by which Pin1 regulates NAFLD, as evidenced by the inhibition of the AMPK/ACC1 pathway. In addition, immunofluorescence (IF), coimmunoprecipitation (Co-IP) and GST pull-down experiments also showed that Pin1 interacts directly with ACC1 and inhibits ACC1 phosphorylation levels. Our study suggests that Pin1 promotes NAFLD progression by inhibiting the activation of the AMPK/ACC1 signalling pathway, and it is possible that this effect is achieved by Pin1 interacting with ACC1 and inhibiting the phosphorylation of ACC1.

Yellow leaf green tea modulates the AMPK/ACC/SREBP1c signaling pathway and gut microbiota in high-fat diet-induced mice to alleviate obesity.

BACKGROUND: Yellow leaf green tea (YLGT) is a new variety of Camellia sinensis (L.) O. Ktze, which has yellow leaves and the unique qualities of 'three green through three yellow'. The present study aimed to investigate the anti-obesity effect of YLGT in mice fed a high-fat diet (HFD) and to explore the potential mechanisms by regulating the AMPK/ACC/SREBP1c signaling pathways and gut microbiota. RESULTS: The results showed that YLGT aqueous extract reduced body weight, hepatic inflammation, fat accumulation and hyperlipidemia in HFD-induced C57BL/6J mice, and also accelerated energy metabolism, reduced fat synthesis and suppressed obesity by activating the AMPK/CPT-1α signaling pathway and inhibiting the FAS/ACC/SREBP-1c signaling pathway. Fecal microbiota transplantation experiment further confirmed that the alteration of gut microbiota (e.g. increasing unclassified_Muribaculaceae and decreasing Colidextribacter) might be an important cause of YLGT water extract inhibiting obesity. CONCLUSION: In conclusion, YLGT has a broad application prospect in the treatment of obesity and the development of anti-obesity function beverages. © 2024 Society of Chemical Industry.