Hexokinase Research Articles

Location matters: hexokinase 1 in glucose metabolism and inflammation

Hexokinase (HK)-1 mitochondrial-binding mechanisms and consequential physiological relevance remain unclear. Recently, De Jesus et al. studied myeloid cells with HK1 carrying mutated mitochondrial-binding domains (MBDs) and provided evidence that HK1 localization controls glucose metabolic fate. Increases in cytosolic HK1 may also contribute to the inflammation associated with diabetes and aging.

OsHXK3 encodes a hexokinase-like protein that positively regulates grain size in rice.

We report the map-based cloning and functional characterization of SNG1, which encodes OsHXK3, a hexokinase-like protein that plays a pivotal role in controlling grain size in rice. Grain size is an important agronomic trait determining grain yield and appearance quality in rice. Here, we report the discovery of rice mutant short and narrow grain1 (sng1) with reduced grain length, width and weight. Map-based cloning revealed that the mutant phenotype was caused by loss of function of gene OsHXK3 that encodes a hexokinase-like (HKL) protein. OsHXK3 was associated with the mitochondria and was ubiquitously distributed in various organs, predominately in younger organs. Analysis of glucose (Glc) phosphorylation activities in young panicles and protoplasts showed that OsHXK3 was a non-catalytic hexokinase (HXK). Overexpression of OsHXK3 could not complement the Arabidopsis glucose insensitive2-1 (gin2-1) mutant, indicating that OsHXK3 lacked Glc signaling activity. Scanning electron microscopy analysis revealed that OsHXK3 affects grain size by promoting spikelet husk cell expansion. Knockout of other nine OsHXK genes except OsHXK3 individually did not change grain size, indicating that functions of OsHXKs have differentiated in rice. OsHXK3 influences gibberellin (GA) biosynthesis and homeostasis. Compared with wild type, OsGA3ox2 was significantly up-regulated and OsGA2ox1 was significantly down-regulated in young panicle of sng1, and concentrations of biologically active GAs were significantly decreased in young panicles of the mutants. The yield per plant of OsHXK3 overexpression lines (OE-4 and OE-35) was increased by 10.91% and 7.62%, respectively, compared to that of wild type. Our results provide evidence that an HXK lacking catalytic and sensory functions plays an important role in grain size and has the potential to increase yield in rice.

Molecular and biochemical effects on metabolism and immunity of Hyriopsis cumingii fed with four different microalgae

Hyriopsis cumingii has attracted attention because of its pearl production performance and water purification capacity. Realizing sustainable industrialized culture of H. cumingii or applying it to bivalve biomanipulation for controlling water eutrophication needs urgent studies about the selection of suitable algae and the effects of different microalgae on mussel physiology. To contrast molecular and biochemical effects of high-quality microalgal diets (Chlorella vulgaris, Navicula pelliculosa, and Cyclotella sp.) with toxic Microcystis aeruginosa on metabolism and immune physiology of H. cumingii, levels of related enzymes and genes were analyzed during the 28-day exposure period. Results showed that the Cyclotella sp. diet could significantly (p < 0.05) maintain higher levels of metabolic enzymes (glutamic oxaloacetate transaminase (GOT), glutamic pyruvate transaminase (GPT), pyruvate kinase (PK), and hexokinase (HK)) and genes (CPT1 and LDLR). C. vulgaris and N. pelliculosa treatments significantly (p < 0.05) reduced activities of these metabolic parameters. The M. aeruginosa treatment significantly (p < 0.05) enhanced levels of immune enzymes (alkaline phosphatase (AKP), superoxide dismutase (SOD), and catalase (CAT)) and genes (HcIL-17 and IAP) on day 1 or 7, and there was a significant (p < 0.05) reduction on day 28. Results suggested that Cyclotella sp. was the suitable algae for H. cumingii, followed by C. vulgaris and N. pelliculosa, and toxic algae caused metabolic disorders, immune injury, and poor physiological status. The study has practical significance in the sustainable cultivation of H. cumingii and provides a theoretical basis for bivalve biomanipulation in eutrophic water.

Genome-Wide Survey and Expression Analyses of Hexokinase Family in Poplar (Populus trichocarpa)

Hexokinase (HXK) family proteins exert critical roles in catalyzing hexose phosphorylation, sugar sensing, and modulation of plant growth and stress adaptation. Nevertheless, a large amount remains unknown about the molecular profile of HXK enzymes in Populus trichocarpa, a woody model tree species. A genome-wide survey of HXK-encoding genes, including phylogenies, genomic structures, exon/intron organization, chromosomal distribution, and conserved features, was conducted, identifying six putative HXK isogenes (PtHXK1-6) in the Populus genome. The evolutionary tree demonstrated that 135 homologous HXKs between 17 plant species were categorized into four major subfamilies (type A, B, C, and D), clustering one plastidic (PtHXK3) and five mitochondrial PtHXKs grouped into type A and B, respectively. The in silico deduction prompted the presence of the conserved sugar-binding core (motif 4), phosphorylation sites (motif 2 and 3), and adenosine-binding domains (motif 7). The transcriptomic sequencing (RNA-seq) and the quantitative real-time PCR (qRT-PCR) assays revealed that three isogenes (PtHXK2, 3, and 6) were abundantly expressed in leaves, stems, and roots, while others appeared to be dominantly expressed in the reproductive tissues. Under the stress exposure, PtHXK2 and 6 displayed a significant induction upon the pathogenic fungi (Fusarium solani) infection and marked promotions by glucose feeding in roots. In contrast, the PtHXK3 and 6 are ABA-responsive genes, following a dose-dependent manner. The comprehensive analyses of the genomic patterns and expression profiling provide theoretical clues and lay a foundation for unraveling the physiological and signaling roles underlying the fine-tuned PtHXKs responding to diverse stressors.

Phosphorylation and acetylation of glycolytic enzymes cooperatively regulate their activity and lamb meat quality

This study examined cooperative regulation of phosphorylation and acetylation of glycolytic enzymes on their activity and lamb meat quality. Muscle samples were divided into two groups (fast and slow) according to their glycolysis rate as defined by pH decline rate from 1 h to 1 d postmortem. In slow glycolysis rate group, the activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) was lower and meat sample showed lower a*, higher shear force and cooking loss. The acetylation and phosphorylation of HK were positively correlated with HK activity. The acetylation and phosphorylation of PFK were correlated with shear force and negatively associated with PFK activity. The acetylation and phosphorylation of PK were significantly correlated with each other but showed insignificant correlations with PK activity. Briefly, the phosphorylation and acetylation of HK, PFK and PK coregulate glycolysis through different crosstalk patterns on their activity and this might affect meat quality.

Huangqin Decoction Exerts Beneficial Effects on Rotenone-Induced Rat Model of Parkinson's Disease by Improving Mitochondrial Dysfunction and Alleviating Metabolic Abnormality of Mitochondria.

Parkinson's disease (PD) is a common neurodegenerative disease, and the pathogenesis of PD is closely related to mitochondrial dysfunction. Previous studies have indicated that traditional Chinese medicine composition of Huangqin Decoction (HQD), including Scutellariae Radix, licorice, and Paeoniae Radix Alba, has therapeutic effects on PD, but whether HQD has a therapeutic effect on PD has not been reported. In this study, the protective effects of HQD on rotenone-induced PD rats were evaluated by behavioral assays (open field, rotating rod, suspension, gait, inclined plate, and grid) and immunohistochemistry. The mechanisms of HQD on attenuation of mitochondrial dysfunction were detected by biochemical assays and mitochondrial metabolomics. The results showed that HQD (20 g/kg) can protect rats with PD by improving motor coordination and muscle strength, increasing the number of tyrosine hydroxylase (TH)-positive neurons in rats with PD. Besides, HQD can improve mitochondrial dysfunction by increasing the content of adenosine triphosphate (ATP) and mitochondrial complex I. Mitochondrial metabolomics analysis revealed that the ketone body of acetoacetic acid (AcAc) in the rotenone group was significantly higher than that of the control group. Ketone bodies have been known to be used as an alternative energy source to provide energy to the brain when glucose was deficient. Further studies demonstrated that HQD could increase the expression of glucose transporter GLUT1, the content of tricarboxylic acid cycle rate-limiting enzyme citrate synthase (CS), and the level of hexokinase (HK) in rats with PD but could decrease the content of ketone bodies [AcAc and β-hydroxybutyric acid (β-HB)] and the expression of their transporters (MCT1). Our study revealed that the decrease of glucose metabolism in the rotenone group was parallel to the increase of substitute substrates (ketone bodies) and related transporters, and HQD could improve PD symptoms by activating the aerobic glycolysis pathway.

Comparative proteomic analysis reveals insights into the response of Cryptolaemus montrouzieri to bottom-up transfer of cadmium and lead across a multi-trophic food chain

Contamination of agro-ecosystems with heavy metals can affect the development and reproduction of insect natural enemies. This study reports a detailed Tandem Mass Tag based quantitative proteomic analysis of underlying mechanisms responsible for stress response of Cryptolaemus montrouzieri against heavy metals (cadmium (Cd) and lead (Pb)) transported across a multi-trophic food chain. A total of 6639 proteins were detected under Cd as well as Pb stress. In Pb versus the control cluster, 69 proteins (28 up-regulated and 41 down-regulated) were differentially expressed whereas 268 proteins were differentially expressed under Cd versus the control cluster, having 198 proteins up-regulated and 70 down-regulated proteins. The analysis of differentially expressed proteins showed that 27 proteins overlapped in both clusters representing the core proteome to Pb and Cd stress. The bioinformatics analysis demonstrated that these proteins were mapped to 57 and 99 pathways in Pb versus control and Cd versus control clusters, respectively. The functional classification by COG, GO and KEGG databases showed significant changes in protein expression by C. montrouzieri under Pb and Cd stress. The heavy metal stress (Pb and Cd) induced significant changes in expression of proteins like hexokinase (HK), succinyl-CoA, trypsin like proteins, cysteine proteases, cell division cycle proteins, and yellow gene proteins. The results provide detailed information on the protein expression levels of C. montrouzieri and will serve as basic information for future proteomic studies on heavy metal responses of insect predators within a multi-trophic food chain.

White Spot Syndrome Virus Triggers a Glycolytic Pathway in Shrimp Immune Cells (Hemocytes) to Benefit Its Replication.

White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that inflicts in huge economic losses in shrimp-farming industry. WSSV triggers aerobic glycolysis in shrimp immune cells (hemocytes), but how this virus regulates glycolytic enzymes or pathway is yet to be characterized. Therefore, mRNA levels and activity of four important glycolytic enzymes, Hexokinase (HK), Phosphofructokinase (PFK), Pyruvate kinase (PK), and Lactate dehydrogenase (LDH), were measured in WSSV-infected shrimp hemocytes. Gene expression of HK and PFK, but not LDH or PK, was increased at the viral genome replication stage (12 hpi); furthermore, activity of these enzymes, except HK, was concurrently increased. However, there was no increased enzyme activity at the viral late stage (24 hpi). In vivo dsRNA silencing and glycolysis disruption by 2-DG further confirmed the role of glycolysis in virus replication. Based on tracing studies using stable isotope labeled glucose, glycolysis was activated at the viral genome replication stage, but not at the viral late stage. This study demonstrated that WSSV enhanced glycolysis by activating glycolytic enzyme at the viral genome replication stage, providing energy and biomolecules for virus replication.

Essential oils of Lippia sidoides and Mentha piperita as reducers of stress during the transport of Colossoma macropomum

This study evaluated the use of the essential oils of Lippia sidoides (EOLS) and Mentha piperita (EOMP) as a stress reducer in the transport of Colossoma macropomum. The fish were transported for four hours in plastic bags, comprising the following treatments: a) non - stressed control (NS - not submitted to transport stress), b) stressed control (S - submitted to transport), c) EOLS 10 mg L−1, d) EOLS 20 mg L−1, e) EOMP 20 mg L−1 and f) EOMP 40 mg L−1; each treatment with three repetitions. After transportation, water quality, hormonal, biochemical and enzymatic parameters were analyzed. C. macropomum transported with EOLS 20 mg L−1 showed a significant reduction in cortisol levels compared to fish transported without essential oil. The glycolytic pathway was particularly stimulated in fish transported with EOMP 20 mg L−1, since the values of glycolytic enzymes hexokinase (HK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) were elevated in muscle tissue. The essential oils did not affect the activity of acetylcholinesterase (AChE) in the brain of C. macropomum and, after transport, the fish from all treatments showed a significant reduction (28.2%) of muscular AChE. The concentrations of EOLS 20 mg L−1 and EOMP 20 mg L−1 promoted increased levels of the enzyme glutathione S-transferase (GST) in the brain. It is concluded that the essential oils in the assessed concentrations tend to improve water quality during transport, minimize physiological stress, help maintain energy demand through the breakdown of hepatic glycogen, glucose production and activation of the glycolytic pathway, however, without accumulating lactate. Furthermore, AChE trials demonstrate that EOs may not have neurotoxic effects. Therefore, the concentration of 20 mg L−1 EOLS is recommended for use in the transport of juvenile C. macropomum.

Endogenous glutamine is rate-limiting for anti-CD3 and anti-CD28 induced CD4+ T-cell proliferation and glycolytic activity under hypoxia and normoxia

To meet the demand for energy and biomass, T lymphocytes (T cells) activated to proliferation and clonal expansion, require uptake and metabolism of glucose (Gluc) and the amino acid (AA) glutamine (Gln). Whereas exogenous Gln is converted to glutamate (Glu) by glutaminase (GLS), Gln is also synthesized from the endogenous pool of AA through Glu and activity of glutamine synthase (GS). Most of this knowledge comes from studies on cell cultures under ambient oxygen conditions (normoxia, 21% O2). However, in vivo, antigen induced T-cell activation often occurs under moderately hypoxic (1–4% O2) conditions and at various levels of exogenous nutrients. Here, CD4+ T cells were stimulated for 72 h with antibodies targeting the CD3 and CD28 markers at normoxia and hypoxia (1% O2). This was done in the presence and absence of the GLS and GS inhibitors, Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES) and methionine sulfoximine (MSO) and at various combinations of exogenous Gluc, Gln and pyruvate (Pyr) for the last 12 h of stimulation. We found that T-cell proliferation, viability and levels of endogenous AA were significantly influenced by the availability of exogenous Gln, Gluc and Pyr as well as inhibition of GLS and GS. Moreover, inhibition of GLS and GS and levels of oxygen differentially influenced oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Finally, BPTES-dependent down-regulation of ECAR was associated with reduced hexokinase (HK) activity at both normoxia and hypoxia. Our results demonstrate that Gln availability and metabolism is rate-limiting for CD4+ T-cell activity.

Lactose Concentration in Low-Lactose and Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients by High Sensitivity Enzymatic Method (K-LOLAC), Collaborative Study: Final Action 2020.08.

The AOAC Stakeholder Panel on Strategic Food Analytical Methods issued a call for methods in 2018 for the measurement of lactose in low-lactose and lactose-free products under Standard Method Performance Requirement (SMPR®) 2018.009. Megazyme's Lactose Assay Kit (K-LOLAC) was reviewed and accepted as a First Action Official MethodSM in 2020 (2020.08). A collaborative study was conducted to evaluate the to evaluate the reproducibility of AOAC Official MethodSM2020.08 for the measurement of lactose concentration in low-lactose and lactose-free milk, milk products, and products containing dairy ingredients. Samples are deproteinated and clarified by treatment with Carrez reagents, and then free glucose is removed using a glucose oxidase and catalase treatment system. Quantification of lactose is based on the hydrolytic activity of β-galactosidase, which hydrolyses lactose to glucose and galactose. Any remaining free D-glucose is first measured using a hexokinase (HK)/glucose 6-phosphate dehydrogenase (G-6PDH)/6-phosphogluconate dehydrogenase (6-PGDH) based assay procedure, and then β-galactosidase is added to hydrolyze the lactose in the same reaction vessel with concurrent measurement of the released D-glucose. The samples analyzed included a number of lactose-free and low-lactose milk samples, lactose-free infant formula, lactose-free milkshake, lactose-free adult nutritional drink, lactose-free cream, and lactose-free cheese. All materials had repeatability relative standard deviations (RSDr) <7%. The reproducibility relative standard deviation (RSDR) varied from 3.8 to 14.9% with seven of the 10 test samples having an RSDR of <10%. The Lactose Assay Kit (K-LOLAC) meets the requirements for reproducibility set out under SMPR 2018.009. The Lactose Assay (K-LOLAC) is a robust, simple, and reproducible method for analysis of lactose in foodstuffs and beverages.

Mathematical Modeling Reveals That Sucrose Regulates Leaf Senescence via Dynamic Sugar Signaling Pathways.

Sucrose (Suc) accumulation is one of the key indicators of leaf senescence onset, but little is known about its regulatory role. Here, we found that application of high (120–150 mM) and low levels (60 mM) of Suc to young leaf (YL) and fully expanded leaf (FEL) discs, respectively, decreased chlorophyll content and maximum photosynthetic efficiency. Electrolyte leakage and malondialdehyde levels increased at high Suc concentrations (90–120 mM in YL and 60 and 150 mM in FEL discs). In FEL discs, the senescence-associated gene NtSAG12 showed a gradual increase in expression with increased Suc application; in contrast, in YL discs, NtSAG12 was upregulated with low Suc treatment (60 mM) but downregulated at higher levels of Suc. In YL discs, trehalose-6-phosphate (T6P) accumulated at a low half-maximal effective concentration (EC50) of Suc (1.765 mM). However, T6P levels declined as trehalose 6 phosphate synthase (TPS) content decreased, resulting in the maximum velocity of sucrose non-fermenting-1-related protein kinase (SnRK) and hexokinase (HXK) occurring at higher level of Suc. We therefore speculated that senescence was induced by hexose accumulation. In FEL discs, the EC50 of T6P occurred at a low concentration of Suc (0.9488 mM); T6P levels progressively increased with higher TPS content, which inhibited SnRK activity with a dissociation constant (Kd) of 0.001475 U/g. This confirmed that the T6P–SnRK complex induced senescence in detached FEL discs.

Stable Isotope Tracing Uncovers Reduced γ/β-ATP Turnover and Metabolic Flux Through Mitochondrial-Linked Phosphotransfer Circuits in Aggressive Breast Cancer Cells.

Changes in dynamics of ATP γ- and β-phosphoryl turnover and metabolic flux through phosphotransfer pathways in cancer cells are still unknown. Using 18O phosphometabolite tagging technology, we have discovered phosphotransfer dynamics in three breast cancer cell lines: MCF7 (non-aggressive), MDA-MB-231 (aggressive), and MCF10A (control). Contrary to high intracellular ATP levels, the 18O labeling method revealed a decreased γ- and β-ATP turnover in both breast cancer cells, compared to control. Lower β-ATP[18O] turnover indicates decreased adenylate kinase (AK) flux. Aggressive cancer cells had also reduced fluxes through hexokinase (HK) G-6-P[18O], creatine kinase (CK) [CrP[18O], and mitochondrial G-3-P[18O] substrate shuttle. Decreased CK metabolic flux was linked to the downregulation of mitochondrial MTCK1A in breast cancer cells. Despite the decreased overall phosphoryl flux, overexpression of HK2, AK2, and AK6 isoforms within cell compartments could promote aggressive breast cancer growth.

Investigation of sugar signaling behaviors involved in sucrose-induced senescence initiation and progression in N. tabacum

Sugar is involved in initiating leaf senescence. However, its regulatory role, especially as a signal in the senescence process, is unclear. Therefore, this study was designed to illustrate how sugar stimulates the onset of leaf senescence and controls sugar homeostasis through the T6P-SnRK (sucrose non-fermenting (SNF)-related kinase) and HXK (hexokinase) signaling pathways. We used a leaf disc system detached from fully expanded leaves of Nicotiana tabacum cv. K326 and designed a time-course study (days 3, 5, 7, and 9) with exogenously gradient concentrations (0, 30, 60, 90, 120, and 150 mM) of sucrose (Suc) treatment to identify how Suc application affects sugar metabolism and induces senescence. Our results revealed that early decreases of Fv/Fm and increases in electrolyte leakage responded to Suc on day 3. Furthermore, a substantial increase in lipid peroxidation and up-regulated expression of senescence marker genes (NtSAG12) (except 60 mM on day 3) responded sequentially by day 5. The glucose, G6P, and HXK contents were first induced by Suc on day 3 and then repressed from day 5 to day 7. However, exogenous Suc treatment significantly improved the TPS content and the subsequent precursor T6P from day 3 to day 7. Following exogenous Suc treatments, the transcript level of NtSnRK1 was markedly down-regulated from day 3 to day 7. On the other hand, a linear regression analysis demonstrated that the T6P-NtSnRK1 signaling pathway was strongly associated with senescence initiation, and was accompanied by membrane degradation and NtCP1/NtSAG12 up-regulation by day 3. The T6P-NtSnRK1 signaling pathway experienced membrane and chloroplast degradation by day 5. HXK functioned as a metabolic enzyme promoting Glc-G6P and as a Glc sensor, accelerating the initiation of senescence through the HXK-dependent pathway by repressing PSII by day 3 and the senescence process through the Glycolytic pathway by day 7. These physiological, biochemical, and molecular analyses demonstrate that exogenous Suc regulates T6P accumulation, inducing senescence through the NtSnRK signaling pathway. These results illustrate the role of Suc and the transition of the sugar signaling pathway during the progression of senescence initiation.

Protein metabolism and immune response levels in cuttlefish (Sepia pharaonis) and the effects of inking stress on development

Inking is a defensive behavior that protects cephalopods from predator attack, a process that is accomplished by generating jet propulsion from powerful contractions of the mantle muscle. However, most studies have focused on the “defensive effects” of the ink, with the result that there is limited information concerning the effects of inking on the animals themselves. On the basis of our previous research, we integrated information from different tissues after continuous ink release to develop a better understanding of the physiological stress resulting from inking in the cuttlefish (Sepia pharaonis). The results showed that throughout the recovery period (60 days) of the inking treatment, the observed declines in growth performance due to decreased protein synthesis and increased energy metabolism levels were supported by a decrease in mantle muscle glycogen concentration (P < 0.01), increased hexokinase (HK) and pyruvate kinase (PK) specifical activities (P < 0.001), up-regulated expression of arginine kinase (AK) and octopine dehydrogenase (ODH) (P < 0.001), and up-regulated immune superoxide dismutase (SOD) and heat shock protein 70 (HSP70) in the digestive gland (P < 0.001). Muscle principal component analysis (PCA) revealed that amino acids (lysine, tyrosinase, arginine, leucine, isoleucine, and aspartate) were involved in protein turnover after the ink release, and essential fatty acids such as oleic acid (18:1n-9), linoleic acid (18:2n-6), arachidonic acid (ARA, 20:4n-6), and docosahexaenoic acid (DHA, 22:6n-3) were disturbed by inking stress. Moreover, the inducible HSP70 in the digestive gland was sensitively up-regulated even after the 60-day recovery, and thus the transcription level of induced HSP70 in cuttlefish may be used as an indicator of physiological stress. In conclusion, a high growth rate requires a high level of protein synthesis, and it appears that considerable ink release has a significant impact on cuttlefish (S. pharaonis) physiological stress. The transcription level of HSP70 may be used as a stress indicator

The hexokinase Gene Family in Cotton: Genome-Wide Characterization and Bioinformatics Analysis.

Hexokinase (HXK) is involved in hexose phosphorylation, sugar sensing, and signal transduction, all of which regulate plant growth and adaptation to stresses. Gossypium hirsutum L. is one of the most important fiber crops in the world, however, little is known about the HXKs gene family in G. hirsutum L. We identified 17 GhHXKs from the allotetraploid G. hirsutum L. genome (AADD). G. raimondii (DD) and G. arboreum (AA) are the diploid progenitors of G. hirsutum L. and contributed equally to the At_genome and Dt_genome GhHXKs genes. The chromosomal locations and exon-intron structures of GhHXK genes among cotton species are conservative. Phylogenetic analysis grouped the HXK proteins into four and three groups based on whether they were monocotyledons and dicotyledons, respectively. Duplication event analysis demonstrated that HXKs in G. hirsutum L. primarily originated from segmental duplication, which prior to diploid hybridization. Experiments of qRT-PCR, transcriptome and promoter cis-elements demonstrated that GhHXKs’ promoters have auxin and GA responsive elements that are highly expressed in the fiber initiation and elongation stages, while the promoters contain ABA-, MeJA-, and SA-responsive elements that are highly expressed during the synthesis of the secondary cell wall. We performed a comprehensive analysis of the GhHXK gene family is a vital fiber crop, which lays the foundation for future studies assessing its role in fiber development.

Follicular fluid-derived exosomal miR-143-3p/miR-155-5p regulate follicular dysplasia by modulating glycolysis in granulosa cells in polycystic ovary syndrome

ObjectivePolycystic ovary syndrome (PCOS) is characterized by follicular dysplasia. An insufficient glycolysis-derived energy supply of granulosa cells (GCs) is an important cause of follicular dysplasia in PCOS. Follicular fluid (FF) exosomal microRNAs (miRNAs) have been proven to regulate the function of GCs. In this study, exosomes extracted from clinical FF samples were used for transcriptome sequencing (RNA-seq) analysis, and a human ovarian granulocyte tumour cell line (KGN cells) was used for in vitro mechanistic studies.Methods and resultsIn FF exosomal RNA-seq analysis, a decrease in glycolysis-related pathways was identified as an important feature of the PCOS group, and the differentially expressed miR-143-3p and miR-155-5p may be regulatory factors of glycolysis. By determining the effects of miR-143-3p and miR-155-5p on hexokinase (HK) 2, pyruvate kinase muscle isozyme M2 (PKM2), lactate dehydrogenase A (LDHA), pyruvate, lactate and apoptosis in KGN cells, we found that upregulated miR-143-3p expression in exosomes from the PCOS group inhibited glycolysis in KGN cells; knockdown of miR-143-3p significantly alleviated the decrease in glycolysis in KGN cells in PCOS. MiR-155-5p silencing attenuated glycolytic activation in KGN cells; overexpression of miR-155-5p significantly promoted glycolysis in KGN cells in PCOS. In this study, HK2 was found to be the mediator of miR-143-3p and miR-155-5p in FF-derived exosome-mediated regulation of glycolysis in KGN cells. Reduced glycolysis accelerated apoptosis of KGN cells, which mediated follicular dysplasia through ATP, lactate and apoptotic pathways.ConclusionsIn conclusion, these results indicate that miR-143-3p and miR-155-5p in FF-derived exosomes antagonistically regulate glycolytic-mediated follicular dysplasia of GCs in PCOS.9LsDb7oAvmV44Q9GUxhugtVideo

Study on the HIF-1α regulated by glycolytic pathways and mitochondrial function in yaks of different altitudes during postmortem aging.

The study investigated the glycolysis pathway mediated by hypoxia-inducible factor-1α (HIF-1α) and the mechanism of its regulation. The results indicated that HIF-1α expression initially increased before subsequently decreasing with aging time during postmortem (p < .01). Glucose transporter-1 (GLUT-1), lactate dehydrogenase (LDH), and hexokinase (HK) displayed a similar trend with aging time (p < .01) while pyruvate dehydrogenase kinase 1 (PDK-1) increased gradually within the first 12 hr before decreasing at 24-120 hr. However, after treatment with a HIF-1α inhibitor, no significant differences were observed in the mitochondrial morphology. Furthermore, lactate content decreased, along with LDH, HK, and F0F1-ATP activities as well as GLUT-1 and PDK-1 expression (p < .01). The shear force for all groups also increased during postmortem aging (p < .01), with that of the controls being significantly higher compared with the treatment groups (p < .01). These findings confirmed that, after slaughter, the hypoxic environment within the muscles provided essential conditions for HIF-1α expression, which, in turn, activated the glycolysis pathway by mediating changes in the activities of glycolytic enzymes and mitochondrial function. Moreover, in accelerating glycolysis rate, the expression of HIF-1α further played a negative role in meat tenderization during postmortem aging. This, it was concluded that HIF-1α expression plays a significant role in postmortem yak meat tenderization by regulating the glycolysis pathway. PRATICAL APPLICATIONS: While converting muscle into meat through hypoxic glycolysis during postmortem aging is undeniable, the biochemical mechanism of this process mediated remains quite obscure. However, the meat quality difference which impact muscle regulation mechanism during postmortem aging has not been reported. The study investigated the HIF-1α played a major role in both the glycolytic pathway and as well as meat tenderness during the postmortem aging of yak meat. The glycolysis pathway is mediated by hypoxia-inducible factor-1α (HIF-1α), the mechanism of its regulation, and meat tenderness during the postmortem aging of yak meat.

New non‐canonical function of catalytically dead hexokinase 1 in ovarian cancer

Hexokinases (HKs) have been found amplified in primary cancer cells, with the amplification of HK1 reported commonly in ovarian cancer cells. The function of HKs extends beyond glucose phosphorylation, and HKs serve as regulatory proteins in tumorigenesis. We addressed the non‐canonical roles of HK1 in ovarian cancer cells by examining the effects of the deletions of HK1 and HK2 in TOV‐112D ovarian adenocarcinoma cells. We reverted these effects by re‐introducing wild‐type HK1 and HK2, and we compared the HK1 revertant with the knock‐in of catalytically‐dead HK1 p.D656A. We subjected these cells to a battery of metabolic and proliferation assays and targeted GCxGC‐MS metabolomics. When expressed in vitro, the p.D656A mutant did not manifest a measurable KM and exhibited Vmax lower by almost two orders of magnitude when compared to the wild‐type enzyme. Knock‐in of HK1 p.D656A into HK1 KO cells reverted the HK1 KO‐induced effects in a broad range of assays, including the alamarBlue cell viability assay under low glucose conditions, the NADPH/NADP and NAD/NADH ratios, and glutathione levels in metformin‐treated cells. In the presence of 0.4 mM glucose, the phenotype of HK1 p.D656A knock‐in cells resembled the phenotype of cells with deleted HK2 but not those with deleted HK1 with regards to the levels of TCA intermediates, aspartate and cysteine, and glutamate. Moreover, HK1 deletion increased the levels of branched amino acids, which was completely eliminated by the expression of catalytically‐dead HK1. The HK1 deletion (but not HK2 deletion) suppressed the growth of xenotransplanted ovarian cancer cells, and nearly abolished the tumor growth when the mice were fed the glucose‐free diet. In summary, we provided the evidence that HK1 is involved in the so far unknown glycolysis‐independent moonlighting function and influences metabolism even in the glucose‐free conditions.

Hexokinase 3 enhances myeloid cell survival via non-glycolytic functions

The family of hexokinases (HKs) catalyzes the first step of glycolysis, the ATP-dependent phosphorylation of glucose to glucose-6-phosphate. While HK1 and HK2 are ubiquitously expressed, the less well-studied HK3 is primarily expressed in hematopoietic cells and tissues and is highly upregulated during terminal differentiation of some acute myeloid leukemia (AML) cell line models. Here we show that expression of HK3 is predominantly originating from myeloid cells and that the upregulation of this glycolytic enzyme is not restricted to differentiation of leukemic cells but also occurs during ex vivo myeloid differentiation of healthy CD34+ hematopoietic stem and progenitor cells. Within the hematopoietic system, we show that HK3 is predominantly expressed in cells of myeloid origin. CRISPR/Cas9 mediated gene disruption revealed that loss of HK3 has no effect on glycolytic activity in AML cell lines while knocking out HK2 significantly reduced basal glycolysis and glycolytic capacity. Instead, loss of HK3 but not HK2 led to increased sensitivity to ATRA-induced cell death in AML cell lines. We found that HK3 knockout (HK3-null) AML cells showed an accumulation of reactive oxygen species (ROS) as well as DNA damage during ATRA-induced differentiation. RNA sequencing analysis confirmed pathway enrichment for programmed cell death, oxidative stress, and DNA damage response in HK3-null AML cells. These signatures were confirmed in ATAC sequencing, showing that loss of HK3 leads to changes in chromatin configuration and increases the accessibility of genes involved in apoptosis and stress response. Through isoform-specific pulldowns, we furthermore identified a direct interaction between HK3 and the proapoptotic BCL-2 family member BIM, which has previously been shown to shorten myeloid life span. Our findings provide evidence that HK3 is dispensable for glycolytic activity in AML cells while promoting cell survival, possibly through direct interaction with the BH3-only protein BIM during ATRA-induced neutrophil differentiation.