Hexokinase Research Articles

Current status and progress of research on the ADP-dependent glucokinase gene.

ADP-dependent glucokinase (ADPGK) produces glucose-6-phosphate with adenosine diphosphate (ADP) as the phosphate group donor, in contrast to ATP-dependent hexokinases (HKs). Originally found in archaea, ADPGK is involved in glycolysis. However, its biological function in most eukaryotic organisms is still unclear, and the molecular mechanism of action requires further investigation. This paper provides a concise overview of ADPGK's origin, biological function and clinical application. It aims to furnish scientific information for the diagnosis and treatment of human metabolic diseases, neurological disorders, and malignant tumours, and to suggest new strategies for the development of targeted drugs.

Five Weeks of Sprint Interval Training Improve Muscle Glycolytic Content and Activity But Not Time to Task Failure in Severe-Intensity Exercise.

This study examined the impact of a 5-wk sprint interval training (SIT) intervention on time to task failure (TTF) during severe-intensity constant work rate (CWR) exercise, as well as in glycolytic enzymatic content and activity, and glycogen content. Fourteen active males were randomized into either a SIT group ( n = 8) composed of 15 SIT sessions over 5 wk, or a control group ( n = 6). At pretraining period, participants performed i) ramp incremental test to measure the cardiorespiratory function; ii) CWR cycling TTF at 150% of the power output (PO) at the respiratory compensation point (RCP-PO) with muscle biopsies at rest and immediately following task failure. After 5 wk, the same evaluations were repeated (i.e., exercise intensities matched to current training status), and an additional cycling CWR matched to pretraining 150% RCP-PO was performed only for TTF evaluation. The content and enzymatic activity of glycogen phosphorylase (GPhos), hexokinase (HK), phosphofructokinase (PFK), and lactate dehydrogenase (LDH), as well as the glycogen content, were analyzed. Content of monocarboxylate transporter isoform 4 (MCT4) and muscle buffering capacity were also measured. Despite improvements in total work performed at CWR posttraining, no differences were observed for TTF. The GPhos, HK, PFK, and LDH content and activity, and glycogen content also improved after training only in the SIT group. Furthermore, the MCT4 concentrations and muscle buffering capacity were also improved only for the SIT group. However, no difference in glycogen depletion was observed between groups and time. Five weeks of SIT improved the glycolytic pathway parameters and total work performed; however, glycogen depletion was not altered during CWR severe-intensity exercise, and TTF remained similar.

Impact of hypoxia on glucose metabolism and hypoxia signaling pathways in juvenile horseshoe crabs Tachypleus tridentatus

Marine hypoxia poses a significant challenge in the contemporary marine environment. The horseshoe crab, an ancient benthic marine organism, is confronted with the potential threat of species extinction due to hypoxia, making it an ideal candidate for studying hypoxia tolerance mechanisms. In this experiment, juvenile Tachypleus tridentatus were subjected to a 21-day trial at DO:2 mg/L (hypoxia) and DO:6 mg/L conditions. The experimental timeline included a 14-day exposure phase followed by a 7-day recovery period. Sampling occurred on days 0, 7, 14, and 21, where the period from day 14 to day 21 corresponds to seven days of recuperation. Several enzymatic activities of important proteins throughout this investigation were evaluated, such as succinate dehydrogenase (SDH), phosphofructokinase (PFK), hexokinase (HK), lactate dehydrogenase (LDH), and pyruvate kinase (PK). Concurrently, the relative expression of hexokinase-1 (HK), hypoxia-inducible factor 1-alpha inhibitor (FIH), and hypoxia-inducible factor 1-alpha (HIF-1α), pyruvate dehydrogenase phosphatase (PDH), succinate dehydrogenase assembly factor 4 (SDH), and Glucose-6-phosphatase (G6Pase) were also investigated. These analyses aimed to elucidate alterations in the hypoxia signaling pathway and respiratory energy metabolism. It is revealed that juvenile T. tridentatus initiated the HIF pathway under hypoxic conditions, resulting in an upregulation of HIF-1α and FIH-1 gene expression, which in turn, influenced a shift in metabolic patterns. Particularly, the activity of glycolysis-related enzymes was promoted significantly, including PK, HK, PKF, LDH, and the related HK gene. In contrast, enzymes linked to aerobic respiration, PDH, and SDH, as well as the related PDH and SDH genes, displayed down-regulation, signifying a transition from aerobic to anaerobic metabolism. Additionally, the activity of gluconeogenesis-related enzymes such as PK and G6Pase gene expression were significantly elevated, indicating the activation of gluconeogenesis and glycogenolysis pathways. Consequently, juvenile T. tridentatus demonstrated an adaptive response to hypoxic conditions, marked by changes in respiratory energy metabolism modes and the activation of hypoxia signaling pathways.

Targeting hexokinase 2 for oral cancer therapy: structure-based design and validation of lead compounds.

The pursuit of small molecule inhibitors targeting hexokinase 2 (HK2) has significantly captivated the field of cancer drug discovery. Nevertheless, the creation of selective inhibitors aimed at specific isoforms of hexokinase (HK) remains a formidable challenge. Here, we present a multiple-pharmacophore modeling approach for designing ligands against HK2 with a marked anti-proliferative effect on FaDu and Cal27 oral cancer cell lines. Molecular dynamics (MD) simulations showed that the prototype ligand exhibited a higher affinity towards HK2. Complementing this, we put forth a sustainable synthetic pathway: an environmentally conscious, single-step process facilitated through a direct amidation of the ester with an amine under transition-metal-free conditions with an excellent yield in ambient temperature, followed by a column chromatography avoided separation technique of the identified lead bioactive compound (H2) that exhibited cell cycle arrest and apoptosis. We observed that the inhibition of HK2 led to the loss of mitochondrial membrane potential and increased mitophagy as a potential mechanism of anticancer action. The lead H2 also reduced the growth of spheroids. Collectively, these results indicated the proof-of-concept for the prototypical lead towards HK2 inhibition with anti-cancer potential.

Real-time resolution studies of the regulation of lactate production by hexokinases binding to mitochondria in single cells.

During hypoxia accumulation of lactate may be a key factor in acidosis-induced tissue damage. Binding of hexokinase (HK) to the outer membrane of mitochondria may have a protective effect under these conditions. We have investigated the regulation of lactate metabolism by hexokinases (HKs), using HEK293 cells in which the endogenous hexokinases have been knocked down to enable overexpression of wild type and mutant HKs. To assess the real-time changes in intracellular lactate levels the cells were also transfected with a lactate specific FRET probe. In the HKI/HKII double knockdown HEK cells, addition of extracellular pyruvate caused a large and sustained decrease in lactate. Upon inhibition of the mitochondrial electron transfer chain by NaCN this effect was reversed as a rapid increase in lactate developed which was followed by a slow and sustained increase in the continued presence of the inhibitor. Incubation of the HKI/HKII double knockdown HEK cells with the inhibitor of the malic enzyme, ME1*, blocked the delayed accumulation of lactate evoked by NaCN. With replacement by overexpression of HKI or HKII the accumulation of intracellular lactate evoked by NaCN was prevented. Blockage of the pentose phosphate pathway with the inhibitor 6-aminonicotinamide (6-AN) abolished the protective effect of HK expression, with NaCN causing again a sustained increase in lactate. The effect of HK was dependent on HK's catalytic activity and interaction with the mitochondrial outer membrane (MOM). Based on these data we propose that transformation of glucose into G6P by HK activates the pentose phosphate pathway which increases the production of NADPH, which then blocks the activity of the malic enzyme to transform malate into pyruvate and lactate.

Unveiling the mechanism of source-sink rebalancing in cucumber-pumpkin heterografts: the buffering roles of rootstock cotyledon.

Grafting onto pumpkin rootstock is widely applied in cucumber production to improve growth and yield, as well as to overcome soil-borne diseases and enhance resistance to abiotic stresses. In this study, we constructed the cucumber-pumpkin heterografts with the one-cotyledon grafting method, and examined the effects of heterografting on biomass allocation and sugar partitioning, with cucumber and pumpkin self-grafts used as control. Compared with cucumber self-grafts, heterografting onto pumpkin rootstock promoted photosynthesis in cucumber scion, and led to higher sucrose contents in the 1st true leaf (source) and newly emerged leaf (sink). Thereby, the scion part of heterografts accumulated more biomass than cucumber self-grafts. In contrast, when compared to pumpkin self-grafts, grafting with cucumber scion reduced root vigor and biomass but promoted cotyledon growth in pumpkin rootstock. The roots (sink) of heterografts contained less sucrose and hexoses, and showed reduced sucrose synthase (SuSy) and hexokinase (HXK) activities. However, the rootstock cotyledon (source) contained more sucrose and starch, and showed higher activities of HXK, cell-wall invertase (CWIN), and enzymes for starch synthesis and degradation. Furthermore, removal or shade of rootstock cotyledon led to reduced growth of root and scion. Silencing of CmoMEX1a gene in rootstock cotyledon inhibited maltose export and reduced root growth of heterografts. These results indicated that rootstock cotyledon, especially its starch content, played a buffering role in the growth regulation of cucumber-pumpkin heterografts. Taken together, our results provided a major contribution to our understanding of source-sink sugar partitioning and scion-rootstock growth balancing in cucumber-pumpkin heterografts.

Antiobesity and antidiabetes effects of Cyperus rotundus rhizomes presenting protein tyrosine phosphatase, dipeptidyl peptidase 4, metabolic enzymes, stress oxidant and inflammation inhibitory potential

Diabetes is a significant global health concern that increases the vulnerability to various chronic illnesses. In view of this issue, the current research aimed to examine the effects of administering an extract derived from the tubers of Cyperus rotundus L (CrE) on obesity, type 1 diabetes, and liver-kidney toxicity. Through the utilization of HPLC-DAD analysis, it was discovered that the extract contained several components, including quercetin (47.8%), luteolin glucoside (17%), luteolin (7.56%), apigenin-7-glucoside (6.29%), naringinin (4.52%), and seven others. In vitro experiments they have demonstrated that CrE effectively inhibited key digestive enzymes associated with obesity and type 2 diabetes, such as DPP-4, PTP1B, lipase, and α-amylase, as evidenced by their respective IC50 values are about 23, 51,83, and 67 μg/ml respectively. Furthermore, when diabetic rats were administered CrE, the activity of pancreatic enzymes linked to inflammation, namely 5-lipoxygenase (5-LO), hyaluronidase (HAase), and myeloperoxidase (MPO), was significantly suppressed by 48, 41, 75, and 47%, respectively. Moreover, CrE exhibited protective effects on pancreatic β-cells by inhibiting the formation of thiobarbituric acid reactive substances (TBARS) by 65% and the induction of superoxide Dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities by 62, 108, and 112% respectively as compared to diabetic untreated rat. Additionally, CrE significantly inhibited the activities of intestinal, pancreatic, and serum lipase and α-amylase activities. In diabetic rats, CrE administration suppressed glycogen phosphorylase (GP) stimulated glycogen synthase (GS) activities by 45 and 30%; and this increased liver glycogen content by 45%. Furthermore, CrE modulated key hepatic enzymes involved in carbohydrate metabolism, including hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), glucose-6-phosphatase (G6P), and fructose-1,6-bisphosphatase (FBP). Notably, the average food and water intake (AFI and AWI) of diabetic rats treated with CrE was reduced by 15 and 16% respectively as compared to those without any treatment. Therefore, this study demonstrated the effectiveness of Cyperus rotundus tubers in preventing and treating obesity and diabetes.

Biallelic hexokinase 1 (HK1) variants causative of non-spherocytic haemolytic anaemia: A case series with emphasis on the HK1 promoter variant and literature review.

The hexokinase (HK) enzyme plays a key role in red blood cell energy production. Hereditary non-spherocytic haemolytic anaemia (HNSHA) caused by HK deficiency is a rare disorder with only 12 different disease-associated variants identified. Here, we describe the clinical features and genotypes of four previously unreported patients with hexokinase 1 (HK1)-related HNSHA, yielding two novel truncating HK1 variants. The patients' phenotypes varied from mild chronic haemolytic anaemia to severe infantile-onset transfusion-dependent anaemia. Three of the patients had mild haemolytic disease caused by the common HK1 promoter c.-193A>G variant combined with an intragenic HK1 variant, emphasizing the importance of including this promoter variant in the haemolytic disease gene panels. HK activity was normal in a severely affected patient with a homozygous HK1 c.2599C>T, p.(His867Tyr) variant, but the affinity for ATP was reduced, hampering the HK function. In cases of HNSHA, kinetic studies should be considered in the functional studies of HK. We reviewed the literature of previously published patients to provide better insight into this rare disease and add to the understanding of genotype-phenotype correlation.

Diabetes mellitus and Alzheimer's disease: Vacuolar adenosine triphosphatase as a potential link.

Globally, the incidence of diabetes mellitus (DM) and Alzheimer's disease (AD) is increasing year by year, causing a huge economic and social burden, and their pathogenesis and aetiology have been proven to have a certain correlation. In recent years, more and more studies have shown that vacuolar adenosine triphosphatases (v-ATPases) in eukaryotes, which are biomolecules regulating lysosomal acidification and glycolipid metabolism, play a key role in DM and AD. This article describes the role of v-ATPase in DM and AD, including its role in glycolysis, insulin secretion and insulin resistance (IR), as well as its relationship with lysosomal acidification, autophagy and β-amyloid (Aβ). In DM, v-ATPase is involved in the regulation of glucose metabolism and IR. v-ATPase is closely related to glycolysis. On the one hand, v-ATPase affects the rate of glycolysis by affecting the secretion of insulin and changing the activities of key glycolytic enzymes hexokinase (HK) and phosphofructokinase 1 (PFK-1). On the other hand, glucose is the main regulator of this enzyme, and the assembly and activity of v-ATPase depend on glucose, and glucose depletion will lead to its decomposition and inactivation. In addition, v-ATPase can also regulate free fatty acids, thereby improving IR. In AD, v-ATPase can not only improve the abnormal brain energy metabolism by affecting lysosomal acidification and autophagy but also change the deposition of Aβ by affecting the production and degradation of Aβ. Therefore, v-ATPase may be the bridge between DM and AD.

Phosphofructokinase mainly affects glycolysis and influences meat quality in postmortem meat

The aim of this study was to evaluate the comparative effects of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) on glycolysis in postmortem meat. Three specific inhibitors were respectively added to postmortem lamb to produce a HK inhibition (HKI) group, a PFK inhibition (PFKI) group and a PK inhibition (PKI) group. Meat samples that assigned to these three groups and a control (CON) group were stored at 4 °C for 5 d. The results indicated that the activity of HK, PFK and PK were effectively restrained during storage. PFK activity was significantly correlated with lactate content (P < 0.05), but HK and PK activity showed a weak relationship with lactate content (P > 0.05). The myofibrillar fragmentation index of the PFKI and PKI groups were significantly lower than that of the CON and HKI groups (P < 0.05). The degradation levels of troponin T and desmin were the smallest in the PFKI group. The result of the myoglobin spectrum at 540 nm and 580 nm showed a decreasing level of oxymyoglobin after HK, PFK and PK inhibition. In conclusion, PFK showed a more dominant effect on glycolysis than HK and PK in postmortem muscle, which may provide a targeted strategy for meat quality regulation through glycolysis.

Evolved changes in phenotype across skeletal muscles in deer mice native to high altitude.

The cold and hypoxic conditions at high altitude necessitate high metabolic O2 demands to support thermogenesis while hypoxia reduces O2 availability. Skeletal muscles play key roles in thermogenesis, but our appreciation of muscle plasticity and adaptation at high altitude has been hindered by past emphasis on only a small number of muscles. We examined this issue in deer mice (Peromyscus maniculatus). Mice derived from both high-altitude and low-altitude populations were born and raised in captivity and then acclimated as adults to normoxia or hypobaric hypoxia (12 kPa O2 for 6-8 wk). Maximal activities of citrate synthase (CS), cytochrome c oxidase (COX), β-hydroxyacyl-CoA dehydrogenase (HOAD), hexokinase (HK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) were measured in 20 muscles involved in shivering, locomotion, body posture, ventilation, and mastication. Principal components analysis revealed an overall difference in muscle phenotype between populations but no effect of hypoxia acclimation. High-altitude mice had greater activities of mitochondrial enzymes and/or lower activities of PK or LDH across many (but not all) respiratory, limb, core and mastication muscles compared with low-altitude mice. In contrast, chronic hypoxia had very few effects across muscles. Further examination of CS in the gastrocnemius showed that population differences in enzyme activity stemmed from differences in protein abundance and mRNA expression but not from population differences in CS amino acid sequence. Overall, our results suggest that evolved increases in oxidative capacity across many skeletal muscles, at least partially driven by differences in transcriptional regulation, may contribute to high-altitude adaptation in deer mice.NEW & NOTEWORTHY Most previous studies of muscle plasticity and adaptation in high-altitude environments have focused on a very limited number of skeletal muscles. Comparing high-altitude versus low-altitude populations of deer mice, we show that a large number of muscles involved in shivering, locomotion, body posture, ventilation, and mastication exhibit greater mitochondrial enzyme activities in the high-altitude population. Therefore, evolved increases in mitochondrial oxidative capacity across skeletal muscles contribute to high-altitude adaptation.

Value of glucose transport protein 1 expression in detecting lymph node metastasis in patients with colorectal cancer.

There are limited data on the use of glucose transport protein 1 (GLUT-1) expression as a biomarker for predicting lymph node metastasis in patients with colorectal cancer. GLUT-1 and GLUT-3, hexokinase (HK)-II, and hypoxia-induced factor (HIF)-1 expressions may be useful biomarkers for detecting primary tumors and lymph node metastasis when combined with fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography (PET/CT). To evaluate GLUT-1, GLUT-3, HK-II, and HIF-1 expressions as biomarkers for detecting primary tumors and lymph node metastasis with 18F-FDG-PET/CT. This retrospective study included 169 patients with colorectal cancer who underwent colectomy and preoperative 18F-FDG-PET/CT at Chungbuk National University Hospital between January 2009 and May 2012. Two tissue cores from the central and peripheral areas of the tumors were obtained and were examined by a dedicated pathologist, and the expressions of GLUT-1, GLUT-3, HK-II, and HIF-1 were determined using immunohistochemical staining. We analyzed the correlations among their expressions, various clinicopathological factors, and the maximum standardized uptake value (SUVmax) of PET/CT. GLUT-1 was found at the center or periphery of the tumors in 109 (64.5%) of the 169 patients. GLUT-1 positivity was significantly correlated with the SUVmax of the primary tumor and lymph nodes, regardless of the biopsy site (tumor center, P < 0.001 and P = 0.012; tumor periphery, P = 0.030 and P = 0.010, respectively). GLUT-1 positivity and negativity were associated with higher and lower sensitivities of PET/CT, respectively, for the detection of lymph node metastasis, regardless of the biopsy site. GLUT3, HK-II, and HIF-1 expressions were not significantly correlated with the SUVmax of the primary tumor and lymph nodes. GLUT-1 expression was significantly correlated with the SUVmax of 18F-FDG-PET/CT for primary tumors and lymph nodes. Clinicians should consider GLUT-1 expression in preoperative endoscopic biopsy in interpreting PET/CT findings.

ZjHXK5 and ZjHXK6 negatively regulate the sugar metabolism of Ziziphus jujuba Mill.

Hexokinase (HXK) plays a crucial role in plants, catalyzing the phosphorylation of hexose substances, which is one of the key steps in sugar metabolism and energy production. While HXK genes have been well-studied in model plants, the evolutionary and functional characteristics of HXK gene family in jujube is unknow. In this study, the HXK gene family members were identified by bioinformatics methods, the key members regulating glucose metabolism were identified by transcriptome data, and finally the function of the key genes was verified by instantaneous and stable genetic transformation. Our results showed that seven HXK genes were identified in the jujube genome, all of which were predict located in the chloroplast and contain Hexokinase-1 (PF00349) and Hexokinase-2 (PF03727) conserved domains. Most of HXK proteins were transmembrane protein with stable, lipid-soluble, hydrophilic. The secondary structure of ZjHXK proteins main α-helix, and contains two distinct tertiary structure. All ZjHXK genes contain nine exons and eight introns. Predictions of cis-regulatory elements indicate that the promoter region of ZjHXK contains a large number of MeJA responsive elements. Finally, combined with the analysis of the relationship between the expression and glucose metabolism, found that ZjHXK5 and ZjHXK6 may the key genes regulating sugar metabolism. Transient overexpression of ZjHXK5 and ZjHXK6 on jujube, or allogeneic overexpression of ZjHXK5 and ZjHXK6 on tomato would significantly reduce the content of total sugar and various sugar components. Transient silencing of ZjHXK5 and ZjHXK6 genes results in a significant increase in sucrose and total sugar content. Interestingly, the expression of ZjHXK5 and ZjHXK6 were also affected by methyl jasmonate.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

2-Deoxyglucose and hydroxychloroquine HPLC-MS-MS analytical methods and pharmacokinetic interactions after oralco-administration in male rats.

Our previous work has shown a synergistic tumoricidal efficacy of combining the hexokinase (HK) inhibitor 2-deoxyglucose (2-DG) and the autophagy inhibitor chloroquine (CQ) through intraperitoneal injections on HK2-addicted prostate cancers in animal models. The pharmacokinetic (PK) behaviors of these oral drugs after simultaneous oral administration have not been reported. We developed high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS-MS) analytical methods for 2-DG and the clinically favored drug hydroxychloroquine (HCQ) for sera samples. Using a jugular vein-cannulated male rat model with serial blood collection before and after a single gavage dose of each drug alone or in combination, we examined their PK metrics for drug-drug interactions. The data demonstrated a rapid and complete separation of 2-DG from common monosaccharides by HPLC-MS-MS multi-reaction monitoring. Application of the HPLC-MS-MS 2-DG and HCQ methods to sera samples of nine rats showed a peak time (Tmax ) for 2-DG of 0.5 h after 2-DG alone or with HCQ and identical post-peak half-life of approximately 1 h. With a seemingly bi-modal time course for HCQ, the Tmax for HCQ alone (1.2 h) was faster than that for the combination (2 h; p = .017). After combination dosing, the peak concentration (Cmax ) and area under the curve (AUC0-4h ) of 2-DG were decreased by 53.8% (p = .0004) and 53.7% (p = .0001), whereas AUC0-8h for HCQ was decreased by 30.8% (p = .0279) from the respective single dosing. Without changing the mean residence time (MRT0-∞ ) of each drug, the combination affected the apparent volume of distribution (Vd ) and clearance (CL) of 2-DG, and CL for HCQ without affecting its Vd . We observed significant negative PK interactions, probably at the intestinal absorption level, between 2-DG and HCQ taken simultaneously by mouth. Future optimization efforts are warranted for their combination regimen for clinical translation.

Active oxygen generation induced by the glucose sensor TaHXK7-1A decreased the drought resistance of transgenic Arabidopsis and wheat (Triticum aestivum L.)

Improving wheat drought resistance is of great significance for grain production and food security. Hexokinases (HXKs) play a role in sugar signal transduction and are involved in abiotic stress responses in wheat. To clarify the relationship between HXKs and drought stress in wheat, we used the rice active oxygen induction gene OsHXK1 as a reference sequence and the homologously cloned wheat TaHXK7-1A gene. TaHXK7-1A was localized in the nucleus and cell membrane. Under drought stress, over-expression of TaHXK7-1A increased the contents of O2·－ and malondialdehyde (MDA) and significantly up-regulated the respiratory burst oxidative homologue (RBOHs) genes in transgenic Arabidopsis. In addition, the over-expression of TaHXK7-1A inhibited the growth of Arabidopsis seedlings and increased ROS accumulation under 6 % exogenous glucose treatment. Gene silencing of TaHXK7-1 decreased the contents of O2·－ and MDA in wheat leaves under drought stress, and the RBOHs was significantly down-regulated, which improved the drought resistance of wheat. The results of yeast one-hybrid, EMSA, and dual-luciferase assays showed that TabHLH148-5A bound to the E-box motif of the TaHXK7-1A promoter and inhibited the expression of TaHXK7-1A. In addition, yeast two-hybrid and luciferase complementation imaging assays showed that TaHXK7-1A interacted with TaGRF3-4A. These results indicate that the glucose sensor TaHXK7-1A was negatively regulated by TabHLH148-5A, interacted with TaGRF3-4A, and negatively regulated wheat drought resistance by regulating RBOHs expression and inducing ROS production, thus providing a theoretical basis for revealing the molecular mechanism of wheat drought resistance.

Hexokinase-linked glycolytic overload and unscheduled glycolysis in hyperglycemia-induced pathogenesis of insulin resistance, beta-cell glucotoxicity, and diabetic vascular complications.

Hyperglycemia is a risk factor for the development of insulin resistance, beta-cell glucotoxicity, and vascular complications of diabetes. We propose the hypothesis, hexokinase-linked glycolytic overload and unscheduled glycolysis, in explanation. Hexokinases (HKs) catalyze the first step of glucose metabolism. Increased flux of glucose metabolism through glycolysis gated by HKs, when occurring without concomitant increased activity of glycolytic enzymes-unscheduled glycolysis-produces increased levels of glycolytic intermediates with overspill into effector pathways of cell dysfunction and pathogenesis. HK1 is saturated with glucose in euglycemia and, where it is the major HK, provides for basal glycolytic flux without glycolytic overload. HK2 has similar saturation characteristics, except that, in persistent hyperglycemia, it is stabilized to proteolysis by high intracellular glucose concentration, increasing HK activity and initiating glycolytic overload and unscheduled glycolysis. This drives the development of vascular complications of diabetes. Similar HK2-linked unscheduled glycolysis in skeletal muscle and adipose tissue in impaired fasting glucose drives the development of peripheral insulin resistance. Glucokinase (GCK or HK4)-linked glycolytic overload and unscheduled glycolysis occurs in persistent hyperglycemia in hepatocytes and beta-cells, contributing to hepatic insulin resistance and beta-cell glucotoxicity, leading to the development of type 2 diabetes. Downstream effector pathways of HK-linked unscheduled glycolysis are mitochondrial dysfunction and increased reactive oxygen species (ROS) formation; activation of hexosamine, protein kinase c, and dicarbonyl stress pathways; and increased Mlx/Mondo A signaling. Mitochondrial dysfunction and increased ROS was proposed as the initiator of metabolic dysfunction in hyperglycemia, but it is rather one of the multiple downstream effector pathways. Correction of HK2 dysregulation is proposed as a novel therapeutic target. Pharmacotherapy addressing it corrected insulin resistance in overweight and obese subjects in clinical trial. Overall, the damaging effects of hyperglycemia are a consequence of HK-gated increased flux of glucose metabolism without increased glycolytic enzyme activities to accommodate it.

The effect and mechanism of hexokinase-2 on cisplatin resistance in lung cancer cells A549.

Hexokinase (HK) is the first rate-limiting enzyme of glycolysis, which can convert glucose to glucose-6-phosphate. There are several subtypes of HK, including HK2, which is highly expressed in a variety of different tumors and is closely associated with survival. Non-small cell lung cancer (NSCLC) A549 cells with stable overexpression and knockdown of HK2 were obtained by lentivirus transfection. The effects of overexpression and knockdown of HK2 on proliferation, migration, invasion, and glycolytic activity of A549 cells were investigated. The effects on apoptosis were also analyzed using western blot and flow cytometry. In addition, the mitochondria and cytoplasm were separated and the expression of apoptotic proteins was detected by western blot respectively. Upregulation of HK2 could promote glycolysis, cell proliferation, migration, and invasion, which would be inhibited through the knockdown of HK2. HK2 overexpression contributed to cisplatin resistance, whereas HK2 knockdown enhanced cisplatin-induced apoptosis in A549 cells. Overexpression of HK2 can promote the proliferation, migration, invasion, and drug resistance of A549 cells by enhancing aerobic glycolysis and inhibiting apoptosis. Reducing HK2 expression or inhibiting HK2 activity can inhibit glycolysis and induce apoptosis in A549 cells, which is expected to be a potential treatment method for NSCLC.

Effect of glycolysis on water holding capacity during postmortem aging of Jersey cattle-yak meat.

Postmortem muscle moisture loss leads to a decrease in carcass weight and can adversely impact overall meat quality. Therefore, it is critical to investigate water holding capacity (WHC) to enhance meat quality. Current research has primarily focused on examining the correlation between signaling molecules and meat quality in relation to the glycolysis effect on muscle WHC. But there exists a significant knowledge gap regarding the mechanism of WHC in Jersey cattle-yak meat. Jersey cattle-yak meat pH decreased and then increased during postmortem aging. Lactate content, cooking loss, pressing loss, drip loss and centrifuging loss of Jersey cattle-yak meat increased and then decreased during postmortem aging. The glycogen content of Jersey cattle-yak meat was significantly higher than that of yak meat at 6-120 h, being 8.40% higher than that of yak meat at 120 h. The activity of key glycolytic enzymes hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) in Jersey cattle-yak meat was lower than that in yak meat. Correlation analysis showed that Jersey cattle-yak meat WHC was positively correlated with the activity of HK, PK, PFK and LDH. The WHC of Jersey cattle-yak meat was higher than that of Gannan yak meat, and it was significantly positively correlated with the activity of key enzymes of the glycolytic signaling pathway. Therefore, the glycolysis rate can be reduced by inhibiting enzyme activity to improve Jersey cattle-yak meat WHC and meat quality. © 2023 Society of Chemical Industry.

Probing Leishmania donovani Metabolism in 3D Culture: New Perspectives on Drug Targets

Objective: The goal of the research was to investigate Leishmania donovani (L. donovani) metabolism in a 3D culture environment and find possible targets for metabolic drugs. Methods: Different metabolic experiments were performed on two strains of L. donovani (LD1S and LD2B) that were cultivated in 3D spheroids. In order to comprehend the metabolic processes of L. donovani, glucose intake, lactate generation, and lipid utilization were evaluated. Hexokinase (HK) and fatty acid synthase (FAS), two important enzymes implicated in these metabolic pathways, were recognized as potential therapeutic targets. HK and FAS were knocked down in the parasite using CRISPR-Cas9 technology to validate these targets, and the effects on growth and viability were statistically evaluated. Results: In the 3D culture, L. donovani showed a strong growth pattern and significantly increased in diameter over the course of 14 days (p&lt;0.001). When compared to earlier 2D investigations, metabolic tests revealed that 3D cells consumed much more glucose and used lipids (p&lt;0.001). In comparison to wild-type strains, HK and FAS knockdown strains drastically decreased their use of lipids and glucose, respectively (p&lt;0.001). In comparison to the wild-type strains, both knockdown variants demonstrated significantly less growth and vitality (p&lt;0.001). Conclusion: Our results demonstrate the value of 3D culture for investigating the metabolism of L. donovani, and they also identify and verify HK and FAS as prospective therapeutic targets. These discoveries could open the door for the creation of novel treatment approaches to treat L. donovani infection. The parasite's considerable growth and survival decrease when these metabolic pathways are interrupted is supported by statistical evidence, opening the door for future therapeutic development.