The bloodstream form of Trypanosoma brucei displays non-canonical gluconeogenesis.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei.

TGFß macrophage reprogramming: a new dimension of macrophage plasticity.

The August 2023 article in Science Signaling, "TGF-ß uncouples glycolysis and inflammation in macrophages and controls survival during sepsis," challenges the traditional M1/M2 macrophage classification by investigating the impact of transforming growth factor ß on macrophage metabolism and function. Despite its conventional anti-inflammatory role, transforming growth factor ß-treated macrophages exhibit a distinct phenotype marked by heightened glycolysis, suppressed proinflammatory cytokines, and increased coagulation factor expression. The study identifies phosphofructokinase, liver type as a crucial glycolytic enzyme regulated by transforming growth factor ß via the mTOR-c-MYC pathway. Epigenetic changes induced by transforming growth factor ß, such as increased Smad3 activation and reduced proinflammatory transcription factor motif enrichment, contribute to the anti-inflammatory profile. The research extends its implications to sepsis, revealing the role of transforming growth factor ß in exacerbating coagulation and reducing survival in mouse models. This effect involves upregulation of coagulation factor F13A1, dependent on phosphofructokinase, liver type activity and glycolysis in macrophages. Connections to COVID-19 pathology are drawn, as transforming growth factor ß-treated macrophages and SARS-CoV-2 E protein-exposed cells display similar metabolic profiles. Bioinformatic analysis of COVID-19 patient data suggests correlations between myeloid expression of TGFßR1, PFKL, and F13A1 with disease severity. The study challenges the M1/M2 classification, emphasizing the complexity of macrophage responses influenced by transforming growth factor ß, proposing transforming growth factor ß as a potential therapeutic target for conditions like sepsis and COVID-19 where dysregulated coagulation is significant. Overall, the research provides valuable insights into transforming growth factor ß-mediated immunometabolic regulation, paving the way for future investigations and potential therapeutic interventions.

High dietary wheat starch negatively regulated growth performance, glucose and lipid metabolisms, liver and intestinal health of juvenile largemouth bass, Micropterus salmoides.

Largemouth bass (Micropterus salmoides) were fed with three diets containing 6%, 12%, and 18% wheat starch for 70 days to examine their impacts on growth performance, glucose and lipid metabolisms, and liver and intestinal health. The results suggested that the 18% starch group inhibited the growth, and improved the hepatic glycogen content compared with the 6% and 12% starch groups (P < 0.05). High starch significantly improved the activities of glycolysis-related enzymes, hexokinase (HK), glucokinase (GK), phosphofructokinase (PFK), and pyruvate kinase (PK) (P < 0.05); promoted the mRNA expression of glycolysis-related phosphofructokinase (pfk); decreased the activities of gluconeogenesis-related enzymes, pyruvate carboxylase (PC), and phosphoenolpyruvate carboxykinase (PEPCK); and reduced the mRNA expression of gluconeogenesis-related fructose-1,6-bisphosphatase-1(fbp1) (P < 0.05). High starch reduced the hepatic mRNA expressions of bile acid metabolism-related cholesterol hydroxylase (cyp7a1) and small heterodimer partner (shp) (P < 0.05), increased the activity of hepatic fatty acid synthase (FAS) (P < 0.05), and reduced the hepatic mRNA expressions of lipid metabolism-related peroxisome proliferator-activated receptor α (ppar-α) and carnitine palmitoyltransferase 1α (cpt-1α) (P < 0.05). High starch promoted inflammation; significantly reduced the mRNA expressions of anti-inflammatory cytokines transforming growth factor-ß1 (tgf-ß1), interleukin-10 (il-10), and interleukin-11ß (il-11ß); and increased the mRNA expressions of pro-inflammatory cytokine tumor necrosis factor-α (tnf-α), interleukin-1ß (il-1ß), and interleukin-8 (il-8) in the liver and intestinal tract (P < 0.05). Additionally, high starch negatively influenced the intestinal microbiota, with the reduced relative abundance of Trichotes and Actinobacteria and the increased relative abundance of Firmicutes and Proteobacteria. In conclusion, low dietary wheat starch level (6%) was more profitable to the growth and health of M. salmoides, while high dietary starch level (12% and 18%) could regulate the glucose and lipid metabolisms, impair the liver and intestinal health, and thus decrease the growth performance of M. salmoides.

NAT10 mediated ac4C acetylation driven m6A modification via involvement of YTHDC1-LDHA/PFKM regulates glycolysis and promotes osteosarcoma.

The dynamic changes of RNA N6-methyladenosine (m6A) during cancer progression participate in various cellular processes. However, less is known about a possible direct connection between upstream regulator and m6A modification, and therefore affects oncogenic progression. Here, we have identified that a key enzyme in N4-acetylcytidine (ac4C) acetylation NAT10 is highly expressed in human osteosarcoma tissues, and its knockdown enhanced m6A contents and significantly suppressed osteosarcoma cell growth, migration and invasion. Further results revealed that NAT10 silence inhibits mRNA stability and translation of m6A reader protein YTHDC1, and displayed an increase in glucose uptake, a decrease in lactate production and pyruvate content. YTHDC1 recognizes differential m6A sites on key enzymes of glycolysis phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) mRNAs, which suppress glycolysis pathway by increasing mRNA stability of them in an m6A methylation-dependent manner. YTHDC1 partially abrogated the inhibitory effect caused by NAT10 knockdown in tumor models in vivo, lentiviral overexpression of YTHDC1 partially restored the reduced stability of YTHDC1 caused by lentiviral depleting NAT10 at the cellular level. Altogether, we found ac4C driven RNA m6A modification can positively regulate the glycolysis of cancer cells and reveals a previously unrecognized signaling axis of NAT10/ac4C-YTHDC1/m6A-LDHA/PFKM in osteosarcoma. Video Abstract.

Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter.

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

p53-responsive CMBL reprograms glucose metabolism and suppresses cancer development by destabilizing phosphofructokinase PFKP.

Aerobic glycolysis is critical for cancer progression and can be exploited in cancer therapy. Here, we report that the human carboxymethylenebutenolidase homolog (carboxymethylenebutenolidase-like [CMBL]) acts as a tumor suppressor by reprogramming glycolysis in colorectal cancer (CRC). The anti-cancer action of CMBL is mediated through its interactions with the E3 ubiquitin ligase TRIM25 and the glycolytic enzyme phosphofructokinase-1 platelet type (PFKP). Ectopic CMBL enhances TRIM25 binding to PFKP, leading to the ubiquitination and proteasomal degradation of PFKP. Interestingly, CMBL is transcriptionally activated by p53 in response to genotoxic stress, and p53 activation represses glycolysis by promoting PFKP degradation. Remarkably, CMBL deficiency, which impairs p53's ability to inhibit glycolysis, makes tumors more sensitive to a combination therapy involving the glycolysis inhibitor 2-deoxyglucose. Taken together, our study demonstrates that CMBL suppresses CRC growth by inhibiting glycolysis and suggests a potential combination strategy for the treatment of CMBL-deficient CRC.

Glycolytic enzyme PFKFB3 regulates sphingosine 1-phosphate receptor 1 in proangiogenic glomerular endothelial cells under diabetic condition.

Glomerular angiogenesis is a characteristic feature of diabetic nephropathy (DN). Enhanced glycolysis plays a crucial role in angiogenesis. The present study was designed to investigate the role of glycolysis in glomerular endothelial cells (GECs) in a mouse model of DN. Mouse renal cortex and isolated glomerular cells were collected for single-cell and RNA sequencing. Cultured GECs were exposed to high glucose in the presence (proangiogenic) and absence of a vascular sprouting regimen. MicroRNA-590-3p was delivered by lipofectamine in vivo and in vitro. In the present study, a subgroup of GECs with proangiogenic features was identified in diabetic kidneys by using sequencing analyses. In cultured proangiogenic GECs, high glucose increased glycolysis and phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) protein expression, which were inhibited by overexpressing miRNA-590-3p. Mimics of miRNA-590-3p also increased receptor for sphingosine 1-phosphate (S1pR1) expression, an angiogenesis regulator, in proangiogenic GECs challenged with high glucose. Inhibition of PFKFB3 by pharmacological and genetic approaches upregulated S1pR1 protein in vitro. Mimics of miRNA-590-3p significantly reduced migration and angiogenic potential in proangiogenic GECs challenged with high glucose. Ten-week-old type 2 diabetic mice had elevated urinary albumin levels, reduced renal cortex miRNA-590-3p expression, and disarrangement of glomerular endothelial cell fenestration. Overexpressing miRNA-590-3p via perirenal adipose tissue injection restored endothelial cell fenestration and reduced urinary albumin levels in diabetic mice. Therefore, the present study identifies a subgroup of GECs with proangiogenic features in mice with DN. Local administration of miRNA-590-3p mimics reduces glycolytic rate and upregulates S1pR1 protein expression in proangiogenic GECs. The protective effects of miRNA-590-3p provide therapeutic potential in DN treatment.NEW & NOTEWORTHY Proangiogenetic glomerular endothelial cells (GECs) are activated in diabetic nephropathy. High glucose upregulates glycolytic enzyme phosphofructokinase/fructose bisphosphatase 3 (PFKFB3) in proangiogenetic cells. PFKFB3 protects the glomerular filtration barrier by targeting endothelial S1pR1. MiRNA-590-3p restores endothelial cell function and mitigates diabetic nephropathy.

Pyrophosphate as allosteric regulator of ATP-phosphofructokinase in Clostridium thermocellum and other bacteria with ATP- and PPi-phosphofructokinases.

The phosphofructokinase (Pfk) reaction represents one of the key regulatory points in glycolysis. While most organisms encode for Pfks that use ATP as phosphoryl donor, some organisms also encode for PPi-dependent Pfks. Despite this central role, the biochemical characteristics as well as the physiological role of both Pfks is often not known. Clostridium thermocellum is an example of a microorganism that encodes for both Pfks, however, only PPi-Pfk activity has been detected in cell-free extracts and little is known about the regulation and function of both enzymes. In this study, the ATP- and PPi-Pfk of C. thermocellum were purified and biochemically characterized. No allosteric regulators were found for PPi-Pfk amongst common effectors. With fructose-6-P, PPi, fructose-1,6-bisP, and Pi PPi-Pfk showed high specificity (KM < 0.62 mM) and maximum activity (Vmax > 156 U mg-1). In contrast, ATP-Pfk showed much lower affinity (K0.5 of 9.26 mM) and maximum activity (14.5 U mg-1) with fructose-6-P. In addition to ATP, also GTP, UTP and ITP could be used as phosphoryl donors. The catalytic efficiency with GTP was 7-fold higher than with ATP, suggesting that GTP is the preferred substrate. The enzyme was activated by NH4+, and pronounced inhibition was observed with GDP, FBP, PEP, and especially with PPi (Ki of 0.007 mM). Characterization of purified ATP-Pfks originating from eleven different bacteria, encoding for only ATP-Pfk or for both ATP- and PPi-Pfk, identified that PPi inhibition of ATP-Pfks could be a common phenomenon for organisms with a PPi-dependent glycolysis.

The mRNA and protein levels of the glycolytic enzymes lactate dehydrogenase A (LDHA) and phosphofructokinase platelet (PFKP) are good predictors of survival time, recurrence, and risk of death in cervical cancer patients.

INTRODUCTION: Patients with cervical cancer (CC) may experience local recurrence very often after treatment; when only clinical parameters are used, most cases are diagnosed in late stages, which decreases the chance of recovery. Molecular markers can improve the prediction of clinical outcome. Glycolysis is altered in 70% of CCs, so molecular markers of this pathway associated with the aggressiveness of CC can be identified. METHODS: The expression of 14 glycolytic genes was analyzed in 97 CC and 29 healthy cervical tissue (HCT) with microarray; only LDHA and PFKP were validated at the mRNA and protein levels in 36 of those CC samples and in 109 new CC samples, and 31 HCT samples by qRT-PCR, Western blotting, or immunohistochemistry. A replica analysis was performed on 295 CC from The Cancer Genome Atlas (TCGA) database. RESULTS: The protein expression of LDHA and PFKP was associated with poor overall survival [OS: LDHA HR = 4.0 (95% CI = 1.4-11.1); p = 8.0 × 10-3 ; PFKP HR = 3.3 (95% CI = 1.1-10.5); p = 4.0 × 10-2 ] and disease-free survival [DFS: LDHA HR = 4.5 (95% CI = 1.9-10.8); p = 1.0 × 10-3 ; PFKP HR = 3.2 (95% CI = 1.2-8.2); p = 1.8 × 10-2 ] independent of FIGO clinical stage, and the results for mRNA expression were similar. The risk of death was greater in patients with overexpression of both biomarkers than in patients with advanced FIGO stage [HR = 8.1 (95% CI = 2.6-26.1; p = 4.3 × 10-4 ) versus HR = 7 (95% CI 1.6-31.1, p = 1.0 × 10-2 )] and increased exponentially as the expression of LDHA and PFKP increased. CONCLUSIONS: LDHA and PFKP overexpression at the mRNA and protein levels was associated with poor OS and DFS and increased risk of death in CC patients regardless of FIGO stage. The measurement of these two markers could be very useful for evaluating clinical evolution and the risk of death from CC and could facilitate better treatment decision making.

A plasma membrane-associated glycolytic metabolon is functionally coupled to KATP channels in pancreatic α and ß cells from humans and mice.

The ATP-sensitive K+ (KATP) channel is a key regulator of hormone secretion from pancreatic islet endocrine cells. Using direct measurements of KATP channel activity in pancreatic ß cells and the lesser-studied α cells, from both humans and mice, we provide evidence that a glycolytic metabolon locally controls KATP channels on the plasma membrane. The two ATP-consuming enzymes of upper glycolysis, glucokinase and phosphofructokinase, generate ADP that activates KATP. Substrate channeling of fructose 1,6-bisphosphate through the enzymes of lower glycolysis fuels pyruvate kinase, which directly consumes the ADP made by phosphofructokinase to raise ATP/ADP and close the channel. We further show the presence of a plasma membrane-associated NAD+/NADH cycle whereby lactate dehydrogenase is functionally coupled to glyceraldehyde-3-phosphate dehydrogenase. These studies provide direct electrophysiological evidence of a KATP-controlling glycolytic signaling complex and demonstrate its relevance to islet glucose sensing and excitability.

The KLF7/PFKL/ACADL axis modulates cardiac metabolic remodelling during cardiac hypertrophy in male mice.

The main hallmark of myocardial substrate metabolism in cardiac hypertrophy or heart failure is a shift from fatty acid oxidation to greater reliance on glycolysis. However, the close correlation between glycolysis and fatty acid oxidation and underlying mechanism by which causes cardiac pathological remodelling remain unclear. We confirm that KLF7 simultaneously targets the rate-limiting enzyme of glycolysis, phosphofructokinase-1, liver, and long-chain acyl-CoA dehydrogenase, a key enzyme for fatty acid oxidation. Cardiac-specific knockout and overexpression KLF7 induce adult concentric hypertrophy and infant eccentric hypertrophy by regulating glycolysis and fatty acid oxidation fluxes in male mice, respectively. Furthermore, cardiac-specific knockdown phosphofructokinase-1, liver or overexpression long-chain acyl-CoA dehydrogenase partially rescues the cardiac hypertrophy in adult male KLF7 deficient mice. Here we show that the KLF7/PFKL/ACADL axis is a critical regulatory mechanism and may provide insight into viable therapeutic concepts aimed at the modulation of cardiac metabolic balance in hypertrophied and failing heart.

Evaluating the impact of the hexosamine biosynthesis pathway and O-GlcNAcylation on glucose metabolism in bovine granulosa cells.

Granulosa cells (GCs) of ovarian follicles prefer glucose as a metabolic substrate for growth and maturation. Disruption of glucose utilization via the hexosamine biosynthesis pathway (HBP) impairs O-linked N-acetylglucosaminylation (O-GlcNAcylation) and inhibits proliferation of bovine GCs of both small (3-5 mm) and large (>8.5 mm) antral follicles. Knowing that 2-5% of all glucose in cells is utilized via the HBP, the aim of this study was to characterize glucose metabolism in bovine GCs and determine the impact of the HBP and O-GlcNAcylation on metabolic activity. The GCs were initially cultured in serum-containing medium to confluency and then sub-cultured in serum-free medium in 96 well plates (n = 10 ovary pairs). The cells were exposed to vehicle and inhibitors of the HBP and O-GlcNAcylation for 24 h. Extracellular acidification rate (ECAR; an indicator of glycolysis) and oxygen consumption rate (OCR; an indicator of oxidative phosphorylation) of the GCs were measured using a Seahorse xFe96 Analyzer, including the implementation of glycolytic and mitochondrial stress tests. GCs from small antral follicles exhibited overall greater metabolic activity than GCs from large antral follicles as evidenced by increased ECAR and OCR. Inhibition of the HBP and O-GlcNAcylation had no effect on the metabolic activity of GCs from either type of follicle. The glycolytic stress test indicated that GCs from both types of follicles possessed additional glycolytic capacity; but again, inhibition of the HBP and O-GlcNAcylation did not affect this. Interestingly, inhibition of cellular respiration by 2-Deoxy-D-glucose impaired OCR only in GCs from small antral follicles, but exposure to the mitochondrial stress test had no effect. Conversely, in GCs from large antral follicles, oxidative metabolism was impaired by the mitochondrial stress test and was accompanied by a concomitant increase in glycolytic metabolism. Immunodetection of glycolytic enzymes revealed that phosphofructokinase expression is increased in GCs of small antral follicles compared to large follicles. Inhibition of O-GlcNAcylation impaired the expression of hexokinase only in GCs of small antral follicles. Inhibition of O-GlcNAcylation also impaired the expression of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in GCs of both types of follicles, but had no effect on the expression of lactate dehydrogenase. The results indicate that GCs of small antral follicles possess greater aerobic glycolytic capacity than GCs from large antral follicles; but disruption of the HBP and O-GlcNAcylation has little to no impact on metabolic activity.

Analysis of differential proteins between non-capacitated and capacitated boar sperm and verification of the effect of phosphofructokinase on capacitation.

The objective of this study was to analyze the differences in the proteins in non-capacitated and capacitated boar sperm and to identify the functions of the differential proteins and key capacitation proteins of boar sperm before and after capacitation. Transwell chambers were used to separate capacitated sperm proteins using a unique polycarbonate membrane. Meanwhile, isotopic tags for relative and absolute quantification combined with LC‒MS/MS analysis were used for quantitative determination of differential proteins. Through the comparative analysis of different databases, 475 different proteins were identified in non-capacitated sperm and capacitated sperm, of which 303 were significantly upregulated and 172 were significantly downregulated. These differentially-expressed proteins are mainly involved in redox processes, cell biosynthesis processes and cell aromatic compound metabolism biological processes. They also participate in the signaling pathways of phosphorylation, ketone synthesis and degradation, most of which interact to varying degrees. Among these differentially-expressed proteins, phosphofructokinase attracted our attention as a potential capacitated protein. We further verified that phosphofructokinase can promote boar sperm capacitation by immunoblotting.

Glycolytic Pfkp acts as a Lin41 protein kinase to promote endodermal differentiation of embryonic stem cells.

Unveiling the principles governing embryonic stem cell (ESC) differentiation into specific lineages is critical for understanding embryonic development and for stem cell applications in regenerative medicine. Here, we establish an intersection between LIF-Stat3 signaling that is essential for maintaining murine (m) ESCs pluripotency, and the glycolytic enzyme, the platelet isoform of phosphofructokinase (Pfkp). In the pluripotent state, Stat3 transcriptionally suppresses Pfkp in mESCs while manipulating the cells to lift this repression results in differentiation towards the ectodermal lineage. Pfkp exhibits substrate specificity changes to act as a protein kinase, catalyzing serine phosphorylation of the developmental regulator Lin41. Such phosphorylation stabilizes Lin41 by impeding its autoubiquitination and proteasomal degradation, permitting Lin41-mediated binding and destabilization of mRNAs encoding ectodermal specification markers to favor the expression of endodermal specification genes. This provides new insights into the wiring of pluripotency-differentiation circuitry where Pfkp plays a role in germ layer specification during mESC differentiation.

The mechanism of Cry41-related toxin against Myzus persicae based on its interaction with Buchnera-derived ATP-dependent 6-phosphofructokinase.

BACKGROUND: Myzus persicae (Hemiptera: Aphididae) is one of the most notorious pests of many crops worldwide. Most Cry toxins produced by Bacillus thuringiensis show very low toxicity to M. persicae; however, a study showed that Cry41-related toxin had moderate toxic activity against M. persicae. In our previous work, potential Cry41-related toxin-binding proteins in M. persicae were identified, including cathepsin B, calcium-transporting ATPase, and Buchnera-derived ATP-dependent 6-phosphofructokinase (PFKA). Buchnera is an endosymbiont present in almost all aphids and it provides necessary nutrients for aphid growth. This study investigated the role of Buchnera-derived PFKA in Cry41-related toxicity against M. persicae. RESULTS: In this study, recombinant PFKA was expressed and purified, and in vitro assays revealed that PFKA bound to Cry41-related toxin, and Cry41-related toxin at 25 µg ml-1 significantly inhibited the activity of PFKA. In addition, when M. persicae was treated with 30 µg ml-1 of Cry41-related toxin for 24 h, the expression of dnak, a single-copy gene in Buchnera, was significantly decreased, indicating a decrease in the number of Buchnera. CONCLUSION: Our results suggest that Cry41-related toxin interacts with Buchnera-derived PFKA to inhibit its enzymatic activity and likely impair cell viability, resulting in a decrease in the number of Buchnera, and finally leading to M. persicae death. These findings open up new perspectives in our understanding of the mode of action of Cry toxins and are useful in helping improve Cry toxicity for aphid control. © 2023 Society of Chemical Industry.

Granulosa cells from immature follicles exhibit restricted glycolysis and reduced energy production: a dominant problem in polycystic ovary syndrome.

PURPOSE: We hypothesized that immature oocytes are associated with impaired energy production in surrounding granulosa cells (GCs) in polycystic ovary syndrome (PCOS). Thus, this study investigated mitochondrial function, determined expression of glycolytic regulatory enzymes, and measured ATP levels in GCs of PCOS patients. METHODS: GCs were isolated from forty-five PCOS patients and 45 control women. Intracellular concentration of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), the rate of glycolysis, total antioxidant capacity (TAC), activities of catalase (CAT) and superoxide dismutase (SOD), and ATP level were measured in GCs. The gene expression and protein levels of glycolytic enzymes (hexokinase, muscular phosphofructokinase, platelet derived phosphofructokinase, and muscular pyruvate kinase) were determined. Association of GC energy level with oocyte maturation was further validated by measuring glycolysis rate and ATP level in GCs isolated from mature and immature follicles from new set of fifteen PCOS patients and 15 controls. RESULTS: PCOS patients showed higher ROS level, decreased TAC, reduced CAT and SOD activities, and lower Δψm together with reduced expression of key glycolytic enzymes. ATP concentration and biochemical pregnancy were lower in PCOS compared with control group. ATP levels were found to be significantly correlated with ROS and Δψm (r = - 0.624 and r = 0.487, respectively). GCs isolated from immature follicles had significantly lower ATP levels and rate of glycolysis compared with the GCs separated from mature follicles in both PCOS patients and control. CONCLUSION: Declined energy due to the mitochondrial dysfunction and restrained glycolysis in GCs is associated with the immature oocytes and lower biochemical pregnancy in PCOS.

Robust arm and leg muscle adaptation to training despite ACE inhibition: a randomized placebo-controlled trial.

PURPOSE: Angiotensin-converting enzyme (ACE) inhibitor treatment is widely applied, but the fact that plasma ACE activity is a potential determinant of training-induced local muscular adaptability is often neglected. Thus, we investigated the hypothesis that ACE inhibition modulates the response to systematic aerobic exercise training on leg and arm muscular adaptations. METHODS: Healthy, untrained, middle-aged participants (40 ± 7 yrs) completed a randomized, double-blinded, placebo-controlled trial. Participants were randomized to placebo (PLA: CaCO3) or ACE inhibitor (ACEi: enalapril) for 8 weeks and completed a supervised, high-intensity exercise training program. Muscular characteristics in the leg and arm were extensively evaluated pre and post-intervention. RESULTS: Forty-eight participants (nACEi = 23, nPLA = 25) completed the trial. Exercise training compliance was above 99%. After training, citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity were increased in m. vastus lateralis in both groups (all P < 0.05) without statistical differences between them (all time × treatment P > 0.05). In m. deltoideus, citrate synthase maximal activity was upregulated to a greater extent (time × treatment P < 0.05) in PLA (51 [33;69] %) than in ACEi (28 [13;43] %), but the change in 3-hydroxyacyl-CoA dehydrogenase and phosphofructokinase maximal activity was similar between groups. Finally, the training-induced changes in the platelet endothelial cell adhesion molecule-1 protein abundance, a marker of capillary density, were similar in both groups in m. vastus lateralis and m. deltoideus. CONCLUSION: Eight weeks of high-intensity whole-body exercise training improves markers of skeletal muscle mitochondrial oxidative capacity, glycolytic capacity and angiogenesis, with no overall effect of pharmacological ACE inhibition in healthy adults.

A functional loop between YTH domain family protein YTHDF3 mediated m6A modification and phosphofructokinase PFKL in glycolysis of hepatocellular carcinoma.

BACKGROUND & AIMS: N6-methyladenosine (m6A) modification plays a critical role in progression of hepatocellular carcinoma (HCC), and aerobic glycolysis is a hallmark of cancer including HCC. However, the role of YTHDF3, one member of the core readers of the m6A pathway, in aerobic glycolysis and progression of HCC is still unclear. METHODS: Expression levels of YTHDF3 in carcinoma and surrounding tissues of HCC patients were evaluated by immunohistochemistry. Loss and gain-of-function experiments in vitro and in vivo were used to assess the effects of YTHDF3 on HCC cell proliferation, migration and invasion. The role of YTHDF3 in hepatocarcinogenesis was observed in a chemically induced HCC model with Ythdf3-/- mice. Untargeted metabolomics and glucose metabolism phenotype assays were performed to evaluate relationship between YTHDF3 and glucose metabolism. The effect of YTHDF3 on PFKL was assessed by methylated RNA immunoprecipitation assays (MeRIP). Co-immunoprecipitation and immunofluorescence assays were performed to investigate the connection between YTHDF3 and PFKL. RESULTS: We found YTHDF3 expression was greatly upregulated in carcinoma tissues and it was correlated with poor prognosis of HCC patients. Gain-of-function and loss-of-function assays demonstrated YTHDF3 promoted proliferation, migration and invasion of HCC cells in vitro, and YTHDF3 knockdown inhibited xenograft tumor growth and lung metastasis of HCC cells in vivo. YTHDF3 knockout significantly suppressed hepatocarcinogenesis in chemically induced mice model. Mechanistically, YTHDF3 promoted aerobic glycolysis by promoting phosphofructokinase PFKL expression at both mRNA and protein levels. MeRIP assays showed YTHDF3 suppressed PFKL mRNA degradation via m6A modification. Surprisingly, PFKL positively regulated YTHDF3 protein expression, not as a glycolysis rate-limited enzyme, and PFKL knockdown effectively rescued the effects of YTHDF3 overexpression on proliferation, migration and invasion ability of Sk-Hep-1 and HepG2 cells. Notably, co-immunoprecipitation assays demonstrated PFKL interacted with YTHDF3 via EFTUD2, a core subunit of spliceosome involved in pre-mRNA splicing process, and ubiquitination assays showed PFKL could positively regulate YTHDF3 protein expression via inhibiting ubiquitination of YTHDF3 protein by EFTUD2. CONCLUSIONS: our study uncovers the key role of YTHDF3 in HCC, characterizes a positive functional loop between YTHDF3 and phosphofructokinase PFKL in glucose metabolism of HCC, and suggests the connection between pre-mRNA splicing process and m6A modification.

TGF-ß3 induces lactate production in Sertoli cell through inhibiting the Notch pathway.

BACKGROUNDS: In the testis, spermatocytes and spermatids rely on lactate produced by Sertoli cells (SCs) as energy source. Transforming growth factor-beta 3 (TGF-ß3) is one of the generally accepted paracrine regulatory factors of SC-created blood-testis barrier (BTB), yet its role in SC glycolysis and lactate production still remains unclear. OBJECTIVES: To investigate the effect of TGF-ß3 on glycolysis and lactate production in SCs and determine the role of lethal giant larvae 2 (Lgl2) and Notch signaling activity during this process. MATERIALS AND METHODS: Primary cultured rat SCs and TM4 cells were treated with different concentrations of TGF-ß3. In some experiments, cells were transfected with siRNA specifically targeting Lgl2 and then treated with TGF-ß3 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Lactate concentration, glucose and glutamine (Gln) consumption in the culture medium, activity of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glutaminase (Gls), ATP level, oxygen consumption, extracellular acidification, and mitochondrial respiration complex activity were detected using commercial kits. The protein level of Lgl2, LDH, monocarboxylate transporter 4 (MCT4), and activity of Akt, ERK, p38 MAPK, and Notch pathway were detected by Western blot. The stage-specific expression of Jagged1 was examined by immunohistochemistry (IHC) and qPCR after laser capture microdissection. Spermatogenesis in rat testis injected with recombinant Jagged1 (re-Jagged1) was observed by HE staining, and lactate concentration in testis lysate was measured at a different day point after re-Jagged1 treatment. RESULTS: Significant enhancement of lactate concentration was detected in a culture medium of both primary SCs and TM4 cells treated with TGF-ß3 at 3 or 5 ng/ml. Besides, other parameters of glycolysis, that is, glucose and Gln consumption, enzyme activity of PFK, LDH, and Gls displayed different levels of increment in primary SCs and TM4 cells after TGF-ß3 treatment. Mitochondria respiration of SCs was shown to decrease in response to TGF-ß3. Lgl2, MCT4, activity of ERK, and p38 MAPK were up-regulated, whereas Akt and Notch pathway activity were inhibited by TGF-ß3. Silencing of Lgl2 in SCs affected lactate production and attenuated the previous effects of TGF-ß3 on SC glycolysis except for Gln consumption, Gls activity, and activity of Akt, ERK, and p38. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment in SCs antagonized glycolysis suppression caused by Lgl2-silencing. In vivo analysis revealed a stage-specific expression of Jagged1 in contrary with TGF-ß3. Activating Notch signaling by re-Jagged1 resulted in restorable hypospermatogenesis and lowered lactate level in rat testis. CONCLUSION: TGF-ß3 induces lactate production in SC through up-regulating Lgl2, which weakened the Notch signaling activity and intensified glycolysis in SCs. Thus, besides the known function of TGF-ß3 as the BTB regulator, TGF-ß3-Lgl2-Notch may be considered an important pathway controlling SC glycolysis and spermatogenesis.

Ultrasonic treatment decreases Lyophyllum decastes fruiting body browning and affects energy metabolism.

Lyophyllum decastes is a common mushroom that is prone to browning during prolonged storage. In this study, the effects of ultrasonic treatment on metabolic gene expression, enzyme activity, and metabolic compounds related to L. decastes browning were investigated. Treatment of the fruiting body at 35 kHz and 300 W for 10 min reduced the browning index of L. decastes by 21.0 % and increased the L* value by 11.1 %. Ultrasonic treatment of the fruiting body resulted in higher levels of total phenols, flavonoids, and 9 kinds of amino acid with catalase (CAT) and peroxidase (POD) activities maintained at high levels. Higher cytochrome c oxidase (CCO), succinate dehydrogenase (SDH), phosphofructokinase (PFK), and pyruvate kinase (PK) activities may be ascribed to increased antioxidant capacity. Moreover, ultrasonication retained higher adenosine triphosphate (ATP) concentrations with an increased energy charge, while there were lower levels of adenosine diphosphate (ADP) and reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+), respectively. Meanwhile, lower lignin contents were observed, along with retarded polyphenol oxidase (PPO) and lipoxygenase (LOX) activities. Lower PPO activity reduced the fruiting body enzymatic browning rate through decreased expression of LdPpo1, LdPpo2, and LdPpo3 during storage at 4 °C for 16 days. This activity may be used to determine the effectiveness of ultrasonication.