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RNA editing is a feature of RNA maturation resulting in the formation of transcripts whose sequence differs from the genome template. Brain RNA editing may be altered in Alzheimers disease (AD). Here, we analyzed data from 1,865 brain samples covering 9 brain regions from 1,074 unrelated subjects on a transcriptome-wide scale to identify inter-regional differences in RNA editing. We expand the list of known brain editing events by identifying 58,761 previously unreported events. We note that only a small proportion of these editing events are found at the protein level in our proteome-wide validation effort. We also identified the occurrence of editing events associated with AD dementia, neuropathological measures and longitudinal cognitive decline in: SYT11, MCUR1, SOD2, ORAI2, HSDL2, PFKP, and GPRC5B. Thus, we present an extended reference set of brain RNA editing events, identify a subset that are found to be expressed at the protein level, and extend the narrative of transcriptomic perturbation in AD to RNA editing.

Simple Summary: Glucose metabolism was the first metabolic pathway to be discovered and serves a central role across all life by generating energy. Cancer cells hijack glycolysis to promote growth and metastasis. Understanding the molecular basis of glycolytic rewiring in cancers could illuminate new treatment approaches. Glycolysis is the central metabolic pathway across all kingdoms of life. Intensive research efforts have been devoted to understanding the tightly orchestrated processes of converting glucose into energy in health and disease. Our review highlights the advances in knowledge of how metabolic and gene networks are integrated through the precise spatiotemporal compartmentalization of rate-limiting enzymes. We provide an overview of technically innovative approaches that have been applied to study phosphofructokinase-1 (PFK1), which represents the fate-determining step of oxidative glucose metabolism. Specifically, we discuss fast-acting chemical biology and optogenetic tools that have delineated new links between metabolite fluxes and transcriptional reprogramming, which operate together to enact tissue-specific processes. Finally, we discuss how recent paradigm-shifting insights into the fundamental basis of glycolytic regulatory control have shed light on the mechanisms of tumorigenesis and could provide insight into new therapeutic vulnerabilities in cancer. [ABSTRACT FROM AUTHOR]

Many cancer cells, regardless of their tissue origin or genetic landscape, have increased expression or activity of the plasma membrane Na-H exchanger NHE1 and a higher intracellular pH (pHi) compared with untransformed cells. A current perspective that remains to be validated is that increased NHE1 activity and pHi enable a Warburg-like metabolic reprogramming of increased glycolysis and decreased mitochondrial oxidative phosphorylation. We tested this perspective and find it is not accurate for clonal pancreatic and breast cancer cells. Using the pharmacological reagent ethyl isopropyl amiloride (EIPA) to inhibit NHE1 activity and decrease pHi, we observe no change in glycolysis, as indicated by secreted lactate and intracellular pyruvate, despite confirming increased activity of the glycolytic enzyme phosphofructokinase-1 at higher pH. Also, in contrast to predictions, we find a significant decrease in oxidative phosphorylation with EIPA, as indicated by oxygen consumption rate (OCR). Decreased OCR with EIPA is not associated with changes in pathways that fuel oxidative phosphorylation or with mitochondrial membrane potential but occurs with a change in mitochondrial dynamics that includes a significant increase in elongated mitochondrial networks, suggesting increased fusion. These findings conflict with current paradigms on increased pHi inhibiting oxidative phosphorylation and increased oxidative phosphorylation being associated with mitochondrial fusion. Moreover, these findings raise questions on the suggested use of EIPA-like compounds to limit metabolic reprogramming in cancer cells.

One of the malignant transformation hallmarks is metabolism reprogramming, which plays a critical role in the biosynthetic needs of unchecked proliferation, abrogating cell death programs, and immunologic escape. However, the mechanism of the metabolic switch is not fully understood. Here, we found that the S-nitrosoproteomic profile of endogenous nitrogen oxide in ovarian cancer cells targeted multiple components in metabolism processes. Phosphofructokinase (PFKM), one of the most important regulatory enzymes of glycolysis, was S-nitrosylated by nitric oxide synthase NOS1 at Cys351. S-nitrosylation at Cys351 stabilized the tetramer of PFKM, leading to resist negative feedback of downstream metabolic intermediates. The PFKM-C351S mutation decreased the proliferation rate of cultured cancer cells, and reduced tumor growth and metastasis in the mouse xenograft model. These findings indicated that S-nitrosylation at Cys351 of PFKM by NOS1 contributes to the metabolic reprogramming of ovarian cancer cells, highlighting a critical role of endogenous nitrogen oxide on metabolism regulations in tumor progression.

Purpose: Our study explored the effect of long noncoding RNA BBOX1-AS1 on colorectal cancer (CRC) radiosensitivity in vivo and in vitro. Methods: Differentially expressed lncRNAs in CRC were screened using a bioinformatics database and an online prediction website. The expression of BBOX1-AS1 in tissue samples was analyzed via real-time quantitative PCR (RT-qPCR). Subcellular localization of BBOX1-AS1 in CRC cells was analyzed using fluorescence in situ hybridization (FISH). The correlation between BBOX1-AS1 and PFK1 expression levels in CRC tissues was analyzed via Pearson's correlation coefficient. The effect of BBOX1-AS1 on PFK1 stability was investigated using RNA and protein stability testing. RNA Binding Protein Immunoprecipitation (RIP) and RNA pull-down assays were used to confirm the binding of BBOX1-AS1 to PFK1. Results: BBOX1-AS1 was highly expressed in CRC and associated with poor prognosis. Similarly, it was highly expressed in CRC tissues and CRC cell lines. In addition, BBOX1-AS1 promoted the proliferation, invasion, migration, and glycolysis of CRC cells and inhibited apoptosis. RIP and RNA pull-down experiments confirmed that BBOX1-AS1 bound to PFK1. RNA stability and protein stability experiments showed that BBOX1-AS1 affected the stability of PFK1 mRNA and protein. Furthermore, we confirmed that BBOX1-AS1 increased radiation resistance through the regulation of PFK1 expression. Conclusions: BBOX1-AS1 promoted the proliferation, invasion, migration, and glycolysis of CRC cells through stabilization of the expression of PFK1. BBOX1-AS1 also inhibited CRC cell apoptosis and increased radiotherapy resistance in CRC cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers. It thrives in a nutrient-poor environment; however, the mechanisms by which PDAC cells undergo metabolic reprogramming to adapt to metabolic stress are still poorly understood. Here, we show that microRNA-135 is significantly increased in PDAC patient samples compared to adjacent normal tissue. Mechanistically, miR-135 accumulates specifically in response to glutamine deprivation and requires ROS-dependent activation of mutant p53, which directly promotes miR-135 expression. Functionally, we found miR-135 targets phosphofructokinase-1 (PFK1) and inhibits aerobic glycolysis, thereby promoting the utilization of glucose to support the tricarboxylic acid (TCA) cycle. Consistently, miR-135 silencing sensitizes PDAC cells to glutamine deprivation and represses tumor growth in vivo. Together, these results identify a mechanism used by PDAC cells to survive the nutrient-poor tumor microenvironment, and also provide insight regarding the role of mutant p53 and miRNA in pancreatic cancer cell adaptation to metabolic stresses.

Allosteric regulation has traditionally been described by mathematically-complex allosteric rate laws in the form of ratios of polynomials derived from the application of simplifying kinetic assumptions. Alternatively, an approach that explicitly describes all known ligand-binding events requires no simplifying assumptions while allowing for the computation of enzymatic states. Here, we employ such a modeling approach to examine the "catalytic potential" of an enzyme-an enzyme's capacity to catalyze a biochemical reaction. The catalytic potential is the fundamental result of multiple ligand-binding events that represents a "tug of war" among the various regulators and substrates within the network. This formalism allows for the assessment of interacting allosteric enzymes and development of a network-level understanding of regulation. We first define the catalytic potential and use it to characterize the response of three key kinases (hexokinase, phosphofructokinase, and pyruvate kinase) in human red blood cell glycolysis to perturbations in ATP utilization. Next, we examine the sensitivity of the catalytic potential by using existing personalized models, finding that the catalytic potential allows for the identification of subtle but important differences in how individuals respond to such perturbations. Finally, we explore how the catalytic potential can help to elucidate how enzymes work in tandem to maintain a homeostatic state. Taken together, this work provides an interpretation and visualization of the dynamic interactions and network-level effects of interacting allosteric enzymes.

Summary: Freshwater invertebrates, especially molluscs, have recently piqued the interest of researchers as a potential new source of food protein hydrolysates. This study prepared bioactive protein hydrolysates (PPh) from the freshwater snail Paludomas conica and investigated their antioxidant and antidiabetic effects by evaluating biochemical and molecular approaches. The DPPH free radical assay, ABTS scavenging assay, FRAP assay and superoxide scavenging assay investigated the antioxidant effects. The antidiabetic potential was measured in α‐amylase and α‐glucosidase inhibition assays followed by in vivo assay in streptozotocin (STZ)‐induced diabetic animal model and gene expression studies. In all antioxidant experiments, the half‐maximal inhibitory concentration (IC50) of PPh was less than the cut‐off value, 1000 μg/mL. The inhibitory concentration of PPh was 1.89 mg/mL for α‐amylase and 334.70 μg/mL for α‐glucosidase in vitro assay. Furthermore, in the streptozotocin‐induced Wistar albino rats model of six groups (n = 5, body weight 180–20 g, age 6–7 weeks), the administration of PPh 250 mg/kg for diabetic rats a 4 weeks intervention attenuated elevated glucose levels and other diabetes‐related biomarkers (alanine aminotransferase, aspartate aminotransferase, lipid profile, total protein, uric acid). Molecular assay by the qPCR analysis showed a significant (P < 0.05) upregulation of the relative mRNA expression of antioxidant and glucose metabolism‐regulating enzymes‐related genes of superoxide dismutase (SOD‐1), Paraoxonase‐1 (PON1) and Phosphofructokinase‐1 (PFK1). Findings demonstrate that PPh could be used as a potential food source to reduce diabetic complications by regulating gene expression of antioxidant and glycolytic enzymes. [ABSTRACT FROM AUTHOR]

Background: It is expected that the expression of key enzymes of the glycolysis pathway, specially PFK-1, increases tumor cells and so enhances the function of this pathway. The p53 and HIF-1 proteins are regulators of the expression of PFK-1 and LDH enzymes. This study was performed to investigate changes in the expression of PFK-1, LDH-A, p53, and HIF-1α genes to identify metabolic changes in non-small cell lung carcinoma (NSCLC) samples. Methods: A number of 30 tumors and their adjacent normal tissue samples from surgically approved NSCLC patients were used. Total RNA from each tissue was extracted. The changes in mRNA levels of PFK-1M, LDHA, p53, and hypoxia-inducible factor-1 (HIF-1α) genes were evaluated in tumor and normal tissues of all patients using the real-time polymerase chain reaction (PCR) method. Finally, statistical analysis was used to determine significant differences and the relationship between changes in mRNA levels. Results: According to the results, there was no significant difference in the mRNA levels of these genes between tumor and normal tissues. A significant difference in the mRNA level of lactate dehydrogenase A (LDHA) between adenocarcinoma (AdC) and squamous cell carcinoma (SqCC) tumor types was observed (P=0.014). Also, the difference between the mRNA level of LDHA and HIF-1α in metastatic and non-metastatic samples was significant (P=0.035 and P=0.046 respectively). Age and male gender were directly associated with an increased risk of NSCLC. Conclusion: The results of the present study revealed that the increase in mRNA level of PFK-1 and p53 may be involved in NSCLC initiation, and an increase in LDH-A and HIF-1a is associated with a metastatic phenotype. [ABSTRACT FROM AUTHOR]

Despite abundant knowledge of the regulation and biochemistry of glycolytic enzymes, we have limited understanding on how they are spatially organized in the cell. Emerging evidence indicates that nonglycolytic metabolic enzymes regulating diverse pathways can assemble into polymers. We now show tetramer- and substrate-dependent filament assembly by phosphofructokinase-1 (PFK1), which is considered the "gatekeeper" of glycolysis because it catalyzes the step committing glucose to breakdown. Recombinant liver PFK1 (PFKL) isoform, but not platelet PFK1 (PFKP) or muscle PFK1 (PFKM) isoforms, assembles into filaments. Negative-stain electron micrographs reveal that filaments are apolar and made of stacked tetramers oriented with exposed catalytic sites positioned along the edge of the polymer. Electron micrographs and biochemical data with a PFKL/PFKP chimera indicate that the PFKL regulatory domain mediates filament assembly. Quantified live-cell imaging shows dynamic properties of localized PFKL puncta that are enriched at the plasma membrane. These findings reveal a new behavior of a key glycolytic enzyme with insights on spatial organization and isoform-specific glucose metabolism in cells.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an autosomal dominant neurodegenerative disease. This disorder is caused by polyglutamine (polyQ)-containing mutant ataxin-3, which tends to misfold and aggregate in neuron cells. We previously demonstrated a protective function of carbonic anhydrase 8 (CA8) in MJD disease models and a decreased glycolytic activity associated with down-regulated CA8 in a human osteosarcoma (OS) cell model. Given that a reduction in body weight accompanied by gait and balance instability was observed in MJD patients and transgenic (Tg) mice, in this study, we aimed to examine whether metabolic defects are associated with MJD and whether CA8 expression is involved in metabolic dysfunction in MJD. Our data first showed that glucose uptake ability decreases in cells harboring mutant ataxin-3, but increases in cells overexpressing CA8. In addition, the expressions of glucose transporter 3 (GLUT3) and phosphofructokinase-1 (PFK1) were significantly decreased in the presence of mutant ataxin-3. Consistently, immunohistochemistry (IHC) showed that GLUT3 was less expressed in cerebella of aged MJD Tg mice, indicating that the dysfunction of GLUT3 may be associated with late-stage disease. On the other hand, transient down-regulation of CA8 revealed decreased expressions of GLUT3 and PFK1 in HEK293 cells harboring wild-type (WT) ataxin-3, but no further reduction of GLUT3 and PFK1 expressions were observed in HEK293 cells harboring mutant ataxin-3. Moreover, immunoprecipitation (IP) and immunofluorescence (IF) demonstrated that interactions exist between ataxin-3, CA8 and GLUT3 in MJD cellular and Tg models. These lines of evidence suggest that CA8 plays an important role in glucose metabolism and has different impacts on cells with or without mutant ataxin-3. Interestingly, the decreased relative abundance of Firmicutes/Bacteroidetes (F/B) ratio in the feces of aged MJD Tg mice coincided with weight loss and metabolic dysfunction in MJD. Taken together, our results are the first to demonstrate the effects of CA8 on glucose metabolism and its involvement in the metabolic defects in MJD disease. Further investigations will be required to clarify the underlying mechanisms for the metabolic defects associated with MJD. [ABSTRACT FROM AUTHOR]

Background: These studies examined mutual protective relationships among rabbit muscle aldolase, enolase, phosphofructokinase-1 (PFK-1) and LDH from inhibitions by ascorbate (AA). It was proposed earlier that specific inhibitions of PFK-1 and LDH by AA faciltated glycogen storage in resting muscle by inhibiting glycolysis. Materials and Methods: The L-ascorbate (AA), L-ascorbyl dibutyrate (AADB), L-ascorbyl dipalmitate (AADP), L-ascorbyl palmitate (AAP), and L-ascorbyl stearate (AS) are shown in Figure 1 and were obtained from TCI and Alfa Aesar. Unless otherwise stated, all enzymes come from rabbit and all experimental temperatures were 25°C, pH 8.0. Results: Rabbit muscle enolase was examined for its protective effect on other rabbit muscle glycolytic enzymes against inhibitions by ascorbate (AA) and some AA-faty acid derivatives. The IC50 values of enolase by ascorbate (AA) and IC50 values of AA-fatty acid derivatives were compared to estimate inhibition potency. For example, ascorbyl dipalmitate (AADP) was 156 times more inhibitory to enolase than AA. It was previously shown that rabbit muscle aldolase prevented LDH activity loses due to AA inhibition and prevented PFK-1 activity losses due both to dilution and AA inhibition; enolase was found to have the same effects as aldolase. Additionally, PFK-1 prevented enolase and LDH inhibitions by AA. LDH did not prevent enolase or PFK-1 from inhibition by AA. LDH did stimulate enolase activity but not PFK-1 activity. Conclusion: The results suggest that interactions among glycolytic enzyme serve to mutually protect one another from activity losses. The inhibition properties of the AA-fatty acid derivatives are discussed in relation to their possible roles in cancer and diabetes. [ABSTRACT FROM AUTHOR]

Background: Previous studies suggested that glycogen storage was facilitated by ascorbate inhibition of phosphofructokinase-1 (PFK-1) in resting mammalian muscle; these studies showed that purified PFK-1 from fish or chicken muscle has properties similar to PFK-1 from mammalian muscles. Materials and Methods: The enzymes utilized in the assay systems came from Sigma-Aldrich Co. Rabbit (Oryctolagus cuniculus) muscle G-actin (A 2522) was free of aldolase, LDH (EC 1.1.1.28) or AK (EC 2.7.4.3) activity and rabbit muscle aldolase (EC 4.1.2.13) was free of AK (EC 2.7.4.3) and LDH activity. Rabbit muscle PFK-1 (RPFK-1), chicken (Gallus gallus) muscle PFK-1 (CPFK-1) and Pacific red snapper (Lutjanus peru) muscle PFK-1 (FPFK-1) used in these tests were prepared from frozen tissues with modifications of a method. AK, LDH, and aldolase activity were absent in purified FPFK-1 and CPFK-1 preparations. Results: It can be shown that the following enzymes associated with glycolysis are not inhibited by 0.1 M ascorbate under our conditions: rabbit muscle aldolase (EC 4.1.2.13); rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12); rabbit muscle phosphoglucose isomerase (EC 5.3.1.9); rabbit muscle pyruvate kinase (EC 2.7.1.40); yeast hexokinase (EC 2,7.1.1); and yeast 3-phosphoglyceric phosphokinase (EC 2.7.2.3). Rabbit muscle enolase (EC4.2.1.11) is inhibited under our conditions. Conclusion: In summary, FPFK-1 and CPFK-1, possess characteristics and behaviors similar to RPFK-1, e.g., losses of activities due to dilutions and protections of some of these activity losses by rabbit muscle aldolase. These interactions of a mammalian aldolase with fish and a bird PFK-1's suggests a conservative evolutionary relationship among aldolases and PFK-1's. [ABSTRACT FROM AUTHOR]

The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.

In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1–2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney. [ABSTRACT FROM AUTHOR]

Phosphofructokinase-1 (PFK-1) is the most important rate-controlling enzyme for glycolysis in both prokaryotes and eukaryotes. PFK-1 activity is regulated by multiple cellular metabolites, including nucleotides. Intracellular nucleotides are produced through various metabolic processes, including energy metabolism and intracellular signaling pathways. The activity of PFK-1, purified from canine skeletal muscle, was evaluated in the presence of various concentrations of adenine nucleotides to examine the regulation of glucose catabolism in canine skeletal muscle. Although UTP did not inhibit PFK-1 activity to the same extent as ATP, it substituted for ATP as a phosphate donor. cAMP functioned in a similar manner as AMP, as an activating effector of the PFK-1 reaction. ADP activated PFK-1 at low concentrations, but slightly inhibited PFK-1 at higher concentrations. The results suggested that canine PFK-1 had three different binding sites for adenine nucleotides acting as phosphate donors, activating effectors, and inhibitory effectors. Moreover, PFK-1 was shown to be activated by AMP and inhibited by ATP, while UTP and cAMP regulated PFK-1 activity. The results also suggested that ADP binds to the allosteric sites (for ATP and AMP) of PFK-1. Each adenine nucleotide functions as either an activating or inhibitory effector of PFK-1 reaction in canine skeletal muscle. Therefore, intracellular nucleotides may play an important role in regulating glucose metabolism by binding to the allosteric site of the enzyme. [ABSTRACT FROM AUTHOR]

Phosphofructokinase-1 (EC:2.7.1.11, PFK-1) catalyzes the phosphorylation of fructose 6-phosphate to fructose 1,6-bisphosphate using adenosine triphosphate and is a key regulatory enzyme of glycolysis. Mammalian PFK-1 isozymes are composed of three kinds of subunits (PFK-M, -L, and -P), with different properties. It has been suggested that the proportion of PFK-1 subunits in different organs is based on the organ energy metabolism. In this study, we analyzed the activity and subunit composition of canine PFK-1. We found that, in dogs, the skeletal muscle only has PFK-M, the liver mainly has PFK-L, and the brain expresses all of them. The knowledge of the composition of PFK-1 could provide useful information for determination of the differences in glycolysis in various organs of dogs. [ABSTRACT FROM AUTHOR]

Scanning fluctuation correlation spectroscopy (FCS) is an experimental technique capable of measuring particle number concentrations by monitoring spontaneous equilibrium fluctuations in the local concentration of a fluorescent species in a small (femtoliter) subvolume of a sample. The method can be used to detect molecular aggregation for dilute, submicromolar samples by directly "counting particles". We introduce the application of two-photon excitation to scanning FCS and discuss its important advantages for this technique. We demonstrate the capability of measuring particle number concentrations in solution, first with dilute samples of monodisperse 7-nm and 15-nm radius latex spheres, and then with B phycoerythrin. The detection of multiple species in a single sample is shown, using mixtures containing both sphere sizes. The method is then applied to study protein aggregation in solution. We monitor the concentration-dependent association/ dissociation equilibrium for glycogen phosphorylase A and malate dehydrogenase. The measured dissociation constants, 430 nM and 144 nM respectively, are in good agreement with previously published values. In addition, oligomer dissociation induced by pH titration from pH 8 to pH 5.0 is detectable for the enyme phosphofructokinase. The possibility of measuring dissociation kinetics by scanning two-photon FCS is also demonstrated using phosphofructokinase.

Aberrant upregulation of the ubiquitin-specific protease 14 (USP14) has been found in some malignant tumors, including oral squamous cell carcinoma (OSCC). In this study, we further demonstrated that aberrantly overexpressed USP14 was also closely related to adverse clinicopathological features and poor prognosis in patients with OSCC, so we hypothesized that USP14 might act as a tumor-promoting factor during the progression of OSCC. Notably, we originally proved that USP14 is a deubiquitinating enzyme for phosphofructokinase-1 liver type (PFKL), a key rate-limiting enzyme involved in the glycolytic pathway. USP14 interacts with PFKL and enhances its stability through deubiquitination in OSCC cells, which in turn enhances PFKL-mediated glycolytic metabolism and ultimately promote cellular proliferation, migration, and tumorigenesis. In this work, we have also demonstrated for the first time that USP14 is a critical regulator of glycolysis in OSCC and verified a novel mechanism whereby it is involved in tumor metastasis and growth. Collectively, our findings provide novel insights into the tumor-promoting role of USP14 and establish mechanistic foundations for USP14-targeting therapies., (© 2024. The Author(s).)

Targeting C5aR1 modulates the function of infiltrated immune cells including tumor-associated macrophages (TAMs). The gut microbiome plays a pivotal role in colorectal cancer (CRC) tumorigenesis and development through TAM education. However, whether and how the gut flora is involved in C5aR1 inhibition-mediated TAMs remains unclear. Therefore, in this study, genetic deletion of C5ar1 or pharmacological inhibition of C5aR1 with anti-C5aR1 Ab or PMX-53 in the presence or absence of deletion Abs were utilized to verify if and how C5aR1 inhibition regulated TAMs polarization via affecting gut microbiota composition. We found that the therapeutic effects of C5aR1 inhibition on CRC benefited from programming of TAMs toward M1 polarization via driving AKT2-mediated 6-phosphofructokinase muscle type (PFKM) stabilization in a TLR5-dependent manner. Of note, in the further study, we found that C5aR1 inhibition elevated the concentration of serum IL-22 and the mRNA levels of its downstream target genes encoded antimicrobial peptides (AMPs), leading to gut microbiota modulation and flagellin releasement, which contributed to M1 polarization. Our data revealed that high levels of C5aR1 in TAMs predicted poor prognosis. In summary, our study suggested that C5aR1 inhibition reduced CRC growth via resetting M1 by AKT2 activation-mediated PFKM stabilization in a TLR5-dependent manner, which relied on IL-22-regulated gut flora., (© 2024. The Author(s).)

We assessed if leptin, a cytokine hormone known to enhance energy expenditure by promoting lipid and carbohydrate catabolism in response to physiologic stress, might directly regulate cellular glycolysis. A transcriptomic analysis of prolactin cells in the tilapia (Oreochromis mossambicus) pituitary rostral pars distalis (RPD) revealed that recombinant leptin (rtLep) differentially regulates 1,995 genes, in vitro. Machine learning algorithms and clustering analyses show leptin influences numerous cellular gene networks including metabolism; protein processing, transport, and metabolism; cell cycle and the hypoxia response. Leptin stimulates transcript abundance of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (gapdh) in a covariate manner to the hypoxic stress gene network. Orthogonal tests confirm that rtLepA dose-dependently increases gapdh gene expression in the RPD along with transcript abundance of 6-phosphofructo-1-kinase (pfk1), the rate limiting glycolytic enzyme. Functional testing demonstrated that leptin stimulates PFK activity and glycolytic output, while Stattic (a STAT3 blocker) was sufficient to suppress these responses, indicating leptin stimulates glycolysis through a STAT3-dependent mechanism. Leptin also stimulated pfk1 gene expression and lactate production in primary hepatocyte incubations in a similar manner to those shown for the pituitary RPD. This work characterizes a critical metabolic action of leptin to directly stimulate glycolysis across tissue types in a teleost model system, and suggest that leptin may promote energy expenditure, in part, by stimulating glycolysis. These data in a teleost fish, suggest that one of leptin's ancient, highly-conserved functions among vertebrates may be stimulation of glycolysis to facilitate the energetic needs associated with various stressors. [ABSTRACT FROM AUTHOR]

Adapting the energy metabolism state to changing bioenergetic demands is essential for mammalian development accompanying massive cell proliferation and cell differentiation. However, it remains unclear how developing embryos meet the changing bioenergetic demands during the chorioallantoic branching (CB) stage, when the maternal-fetal exchange of gases and nutrients is promoted. In this study, using metabolome analysis with mass-labeled glucose, we found that developing embryos redirected glucose carbon flow into the pentose phosphate pathway via suppression of the key glycolytic enzymes PFK-1 and aldolase during CB. Concomitantly, embryos exhibited an increase in lactate pool size and in the fractional contribution of glycolysis to lactate biosynthesis. Imaging mass spectrometry visualized lactate-rich tissues, such as the dorsal or posterior neural tube, somites and head mesenchyme. Furthermore, we found that the heterochronic gene Lin28a could act as a regulator of the metabolic changes observed during CB. Perturbation of glucose metabolism rewiring by suppressing Lin28a downregulation resulted in perinatal lethality. Thus, our work demonstrates that developing embryos rewire glucose metabolism following CB for normal development. [ABSTRACT FROM AUTHOR]

After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB-treated group and 36.7% decrease in 1 mM 3-AB-treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8± 6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells. [ABSTRACT FROM AUTHOR]

Phosphofructokinase-1 (PFK-1), a major regulatory glycolytic enzyme, has been implicated in the functions of astrocytes and neurons. Here, we report that PFK-1 negatively regulates neurogenesis from neural stem cells (NSCs) by targeting pro-neural transcriptional factors. Using in vitro assays, we found that PFK-1 knockdown enhanced, and PFK-1 overexpression inhibited the neuronal differentiation of NSCs, which was consistent with the findings from NSCs subjected to 5 h of hypoxia. Meanwhile, the neurogenesis induced by PFK-1 knockdown was attributed to the increased proliferation of neural progenitors and the commitment of NSCs to the neuronal lineage. Similarly, in vivo knockdown of PFK-1 also increased neurogenesis in the dentate gyrus of the hippocampus. Finally, we demonstrated that the neurogenesis mediated by PFK-1 was likely achieved by targeting mammalian achaete-scute homologue-1 (Mash 1), neuronal differentiation factor (NeuroD), and sex-determining region Y (SRY)-related HMG box 2 (Sox2). All together, our results reveal PFK-1 as an important regulator of neurogenesis. [ABSTRACT FROM AUTHOR]

Although the influence of diabetes on salivary glands is well studied, it still presents conflicting results. In this work, the regulation of the phosphofructokinase-1 enzyme (PFK-1) was studied utilizing the salivary glands of rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin (60 mg/Kg of body weight) in rats (180-200 g). The animals were killed 30 days after the induction of diabetes and the submandibular and parotid salivary glands were used. Hyperglycemia was evaluated by blood sugar determination. The distribution of PFK-1 between the soluble and cytoskeleton fractions, the phosphate content of PFK-1, the content of fructose-2,6-bisphosphate and the activity of the PFK-2 enzyme were determined. The calculated relative glandular weight showed a higher value for the parotid gland in comparison with the control, but not for the submandibular gland. The activity of PFK-1 expressed per gland showed no variation between diabetic and control animals. However, considering the specific activity, the soluble enzyme presented a value 50% higher than that of the control and the cytoskeleton bound form increased by 84% compared to the control. For the parotid gland, no difference in the specific activity between diabetic and control animals was observed. On the other hand, the activity per gland of the soluble enzyme increased in the diabetic animals. The phosphate content of PFK-1 increased in the submandibular and parotid glands of diabetic rats. Both the content of fructose-2,6-bisphosphate and the active form of PFK-2 were reduced in the diabetic glands. In conclusion, the increase in the activity of PFK-1 observed in the salivary glands of rats with streptozotocin-induced diabetes does not seem to be due to its modulator fructose-2,6-bisphosphate.Apesar de existirem muitos estudos sobre a influência do diabetes nas glândulas salivares, esses apresentam resultados conflitantes. Neste estudo, a regulação da enzima fosfofrutoquinase-1 (PFK-1) foi estudada utilizando-se glândulas salivares de ratos. O diabetes foi induzido por uma única injeção intraperitonial de estreptozotocina (60 mg/kg peso corporal) em ratos (180-200 g). Os animais foram sacrificados 30 dias após a indução do diabetes e utilizaram-se as glândulas submandibular e parótida. A hiperglicemia foi avaliada por determinação da glicemia sanguínea. A distribuição da PFK-1 entre frações solúvel e ligada, concentração de fosfato na PFK-1, concentração de frutose-2,6-bisfosfato e a atividade da enzima PFK-2 foram determinadas. O cálculo do peso glandular relativo mostrou um aumento na glândula parótida de ratos diabéticos comparados ao controle, o que não ocorreu na glândula submandibular. A atividade da PFK-1 expressa por glândula não mostrou variação entre animais diabético e controle. Contudo, considerando a atividade específica, a fração solúvel da enzima mostrou aumento de 50% com relação ao controle e a fração ligada ao citoesqueleto um aumento de 84% com relação ao controle. Na glândula parótida não foi observada diferença na atividade específica entre os grupos diabético e controle. Por outro lado, a atividade por glândula da fração solúvel aumentou nos animais diabéticos. A concentração de fosfato da PFK-1 aumentou nas glândulas submandibular e parótida nos animais diabéticos. Tanto a concentração de frutose-2,6-bisfosfato quanto a forma ativa da PFK-2 mostraram redução nas glândulas salivares. Concluindo, o aumento na atividade da PFK-1 observado nas glândulas salivares de ratos com diabetes induzida por estreptozotocina não parece ser modulado pela frutose-2,6-bisfosfato.