Age-Dependent Changes in the Production of Mitochondrial Reactive Oxygen Species in Human Skeletal Muscle.

A decrease in muscle mass and its functionality (strength, endurance, and insulin sensitivity) is one of the integral signs of aging. One of the triggers of aging is an increase in the production of mitochondrial reactive oxygen species. Our study was the first to examine age-dependent changes in the production of mitochondrial reactive oxygen species related to a decrease in the proportion of mitochondria-associated hexokinase-2 in human skeletal muscle. For this purpose, a biopsy was taken from m. vastus lateralis in 10 young healthy volunteers and 70 patients (26-85 years old) with long-term primary arthrosis of the knee/hip joint. It turned out that aging (comparing different groups of patients), in contrast to inactivity/chronic inflammation (comparing young healthy people and young patients), causes a pronounced increase in peroxide production by isolated mitochondria. This correlated with the age-dependent distribution of hexokinase-2 between mitochondrial and cytosolic fractions, a decrease in the rate of coupled respiration of isolated mitochondria and respiration when stimulated with glucose (a hexokinase substrate). It is discussed that these changes may be caused by an age-dependent decrease in the content of cardiolipin, a potential regulator of the mitochondrial microcompartment containing hexokinase. The results obtained contribute to a deeper understanding of age-related pathogenetic processes in skeletal muscles and open prospects for the search for pharmacological/physiological approaches to the correction of these pathologies.

Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.

Poly(ADP-ribose) mediates bioenergetic defects and redox imbalance in neurons following oxygen and glucose deprivation.

PARP-1 over-activation results in cell death via excessive PAR generation in different cell types, including neurons following brain ischemia. Glycolysis, mitochondrial function, and redox balance are key cellular processes altered in brain ischemia. Studies show that PAR generated after PARP-1 over-activation can bind hexokinase-1 (HK-1) and result in glycolytic defects and subsequent mitochondrial dysfunction. HK-1 is the neuronal hexokinase and catalyzes the first reaction of glycolysis, converting glucose to glucose-6-phosphate (G6P), a common substrate for glycolysis, and the pentose phosphate pathway (PPP). PPP is critical in maintaining NADPH and GSH levels via G6P dehydrogenase activity. Therefore, defects in HK-1 will not only decrease cellular bioenergetics but will also cause redox imbalance due to the depletion of GSH. In brain ischemia, whether PAR-mediated inhibition of HK-1 results in bioenergetics defects and redox imbalance is not known. We used oxygen-glucose deprivation (OGD) in mouse cortical neurons to mimic brain ischemia in neuronal cultures and observed that PARP-1 activation via PAR formation alters glycolysis, mitochondrial function, and redox homeostasis in neurons. We used pharmacological inhibition of PARP-1 and adenoviral-mediated overexpression of wild-type HK-1 (wtHK-1) and PAR-binding mutant HK-1 (pbmHK-1). Our data show that PAR inhibition or overexpression of HK-1 significantly improves glycolysis, mitochondrial function, redox homeostasis, and cell survival in mouse cortical neurons exposed to OGD. These results suggest that PAR binding and inhibition of HK-1 during OGD drive bioenergetic defects in neurons due to inhibition of glycolysis and impairment of mitochondrial function.

Involvement of DJ-1 in the pathogenesis of intervertebral disc degeneration via hexokinase 2-mediated mitophagy.

Intervertebral disc degeneration (IDD) is an important pathological basis for degenerative spinal diseases and is involved in mitophagy dysfunction. However, the molecular mechanisms underlying mitophagy regulation in IDD remain unclear. This study aimed to clarify the role of DJ-1 in regulating mitophagy during IDD pathogenesis. Here, we showed that the mitochondrial localization of DJ-1 in nucleus pulposus cells (NPCs) first increased and then decreased in response to oxidative stress. Subsequently, loss- and gain-of-function experiments revealed that overexpression of DJ-1 in NPCs inhibited oxidative stress-induced mitochondrial dysfunction and mitochondria-dependent apoptosis, whereas knockdown of DJ-1 had the opposite effect. Mechanistically, mitochondrial translocation of DJ-1 promoted the recruitment of hexokinase 2 (HK2) to damaged mitochondria by activating Akt and subsequently Parkin-dependent mitophagy to inhibit oxidative stress-induced apoptosis in NPCs. However, silencing Parkin, reducing mitochondrial recruitment of HK2, or inhibiting Akt activation suppressed DJ-1-mediated mitophagy. Furthermore, overexpression of DJ-1 ameliorated IDD in rats through HK2-mediated mitophagy. Taken together, these findings indicate that DJ-1 promotes HK2-mediated mitophagy under oxidative stress conditions to inhibit mitochondria-dependent apoptosis in NPCs and could be a therapeutic target for IDD.

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.

Targeted degradation of hexokinase 2 for anti­inflammatory treatment in acute lung injury.

Acute lung injury (ALI) is an acute inflammatory lung disease associated with both innate and adaptive immune responses. Hexokinase 2 (HK2) is specifically highly expressed in numerous types of inflammation­related diseases and models. In the present study in vitro and in vivo effects of targeted degradation of HK2 on ALI were explored. The degradation of HK2 by the targeting peptide TAT (transactivator of transcription protein of HIV­1)­ataxin 1 (ATXN1)­chaperone­mediated autophagy­targeting motif (CTM) was demonstrated by ELISA and western blotting in vitro and in vivo. The inhibitory effects of TAT­ATXN1­CTM on lipopolysaccharide (LPS)­induced inflammatory responses were examined using ELISAs. The therapeutic effects of TAT­ATXN1­CTM on LPS­induced ALI were examined via histological examination and ELISAs in mice. 10 µM TAT­ATXN1­CTM administration decreased HK2 protein expression and the secretion of proinflammatory cytokines (TNF­α and IL­1ß) without altering HK2 mRNA expression in LPS­treated both in vitro and in vivo, while pathological lung tissue damage and the accumulation of leukocytes, neutrophils, macrophages and lymphocytes in ALI were also significantly suppressed by 10 µM TAT­ATXN1­CTM treatment. TAT­ATXN1­CTM exhibited anti­inflammatory activity in vitro and decreased the severity of ALI in vivo. HK2 degradation may represent a novel therapeutic approach for ALI.

New insights into the treatment of polycystic ovary syndrome: HKDC1 promotes the growth of ovarian granulocyte cells by regulating mitochondrial function and glycolysis.

Polycystic ovary syndrome (PCOS) is an endocrine disease, and its pathogenesis and treatment are still unclear. Hexokinase domain component 1 (HKDC1) participates in regulating mitochondrial function and glycolysis. However, its role in PCOS development remains unrevealed. Here, female C57BL/6 mice were intraperitoneally injected with dehydroepiandrosterone (DHEA; 60 mg/kg body weight) to establish an in vivo model of PCOS. In vitro, KGN cells, a human ovarian granular cell line, were used to explore the potential mechanisms. DHEA-treated mice exhibited a disrupted estrus cycle, abnormal hormone levels, and insulin resistance. Dysfunction in mitochondria and glycolysis is the main reason for PCOS-related growth inhibition of ovarian granular cells. Here, we found that the structure of mitochondria was impaired, less ATP was generated and more mitochondrial Reactive Oxygen Species were produced in HKDC1-silenced KGN cells. Moreover, HKDC1 knockdown inhibited glucose consumption and decreased the production of glucose-6-phosphate and lactic acid. Conclusively, HKDC1 protects ovarian granulocyte cells from DHEA-related damage at least partly by preserving mitochondrial function and maintaining glycolysis.

Identification of HK3 as a promising immunomodulatory and prognostic target in sepsis-induced acute lung injury.

BACKGROUND: Sepsis is a life-threatening global disease with a significant impact on human health. Acute lung injury (ALI) has been identified as one of the primary causes of mortality in septic patients. This study aimed to identify candidate genes involved in sepsis-induced ALI through a comprehensive approach combining bioinformatics analysis and experimental validation. METHODS: The datasets GSE65682 and GSE32707 obtained from the Gene Expression Omnibus database were merged to screen for sepsis-induced ALI related differentially expressed genes (DEGs). Functional enrichment and immune infiltration analyses were conducted on DGEs, with the construction of protein-protein interaction (PPI) networks to identify hub genes. In vitro and in vivo models of sepsis-induced ALI were used to study the expression and function of hexokinase 3 (HK3) using various techniques including Western blot, real-time PCR, immunohistochemistry, immunofluorescence, Cell Counting Kit-8, Enzyme-linked immunosorbent assay, and flow cytometry. RESULTS: The results of bioinformatics analysis have identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic significance for sepsis-induced ALI. The HK3 has profound effects on sepsis-induced ALI and exhibits a correlation with immune regulation. Experimental results showed increased HK3 expression in lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro studies demonstrated upregulation of HK3 in lipopolysaccharide (LPS)-stimulated lung epithelial cells, with cytoplasmic localization around the nucleus. Interestingly, following the knockdown of HK3 expression, lung epithelial cells exhibited a significant decrease in proliferation activity and glycolytic flux, accompanied by an increase in cellular inflammatory response, oxidative stress, and cell apoptosis. CONCLUSIONS: It was observed for the first time that HK3 plays a crucial role in the progression of sepsis-induced ALI and may be a valuable target for immunomodulation and therapy.Bioinformatics analysis identified HK3, MMP9, and S100A8 as hub genes with diagnostic and prognostic relevance in sepsis-induced ALI. Experimental findings showed increased HK3 expression in the lung tissue of septic mice, particularly in bronchial and alveolar epithelial cells. In vitro experiments demonstrated increased HK3 levels in lung epithelial cells stimulated with LPS, with cytoplasmic localization near the nucleus. Knockdown of HK3 expression resulted in decreased proliferation activity and glycolytic flux, increased inflammatory response, oxidative stress, and cell apoptosis in lung epithelial cells.

LC/MS/MS and GC/MS/MS metabolic profiling of Leontodon hispidulus, in vitro and in silico anticancer activity evaluation targeting hexokinase 2 enzyme.

Leontodon hispidulus Boiss is a wild annual plant growing in Egypt. The present study aims for the first time, to evaluate the phytochemical profile of the main secondary metabolites of the optimized ethanolic extract of the plant using Quadrupole Time-of-Flight Liquid chromatography-mass spectrometry and Gas chromatography-mass spectrometry. It also aims to assess the anticancer activity of its different fractions against the prostate carcinoma cell line. Moreover, an in-silico docking study was performed using the Hexokinase-two enzyme. LC-qToF-MS analysis revealed the tentative identification of 36 phenolic compounds including the glycosides of (luteolin, quercetin, kaempferol, apigenin, isorhamnetin, and daidzein), coumarines (esculin, esculetin, and daphnetin), and phenolic acids (chlorogenic, caffeic, quinic, P-coumaric, and rosmarinic). GC-MS/MS analysis revealed the presence of 18 compounds where palmitic acid, myristic acid, alpha-amyrin, and beta-amyrin were the major ones. The cytotoxic activity results revealed that methylene chloride and ethyl acetate fractions showed the highest cytotoxic activity against the PC3 cell line, with IC50 values of 19, and 19.6 µg/ml, respectively. Interestingly, the docking study demonstrated that apigenin-7-O-glucoside, luteolin-7-O-glucoside, kaempferol-3-O-glucuronide, quercetin-4'-O-glucoside, esculin, rosmarinic acid, chlorogenic acid, and α-amyrin exhibited high affinity to the selected target, HEK-2 enzyme.

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.

A gain of function mutation in AKT1 increases hexokinase 2 and diminishes oxidative stress in meningioma.

Increasing evidence suggests the oncogenic role of missense mutation (AKT1-E17K) of AKT1 gene in meningiomas. Upon investigating the connection between the pro-tumorigenic role of AKT1-E17K and cellular metabolic adaptations, elevated levels of glycolytic enzyme hexokinase 2 (HK2) was observed in meningioma patients with AKT1-E17K compared to patients harboring wild-type AKT1. In vitro experiments also suggested higher HK2 levels and its activity in AKT1-E17K cells. Treatment with the conventional drug of choice AZD5363 (a pan AKT inhibitor) enhanced cell death and diminished HK2 levels in AKT1 mutants. Given the role of AKT phosphorylation in eliciting inflammatory responses, we observed increased levels of inflammatory mediators (IL-1ß, IL6, IL8, and TLR4) in AKT1-E17K cells compared to AKT1-WT cells. Treatment with AKT or HK2 inhibitors dampened the heightened levels of inflammatory markers in AKT1-E17K cells. As AKT and HK2 regulates redox homeostasis, diminished ROS generation concomitant with increased levels of NF-E2- related factor 2 (Nrf2) and superoxide dismutase 1 (SOD1) were observed in AKT1-E17K cells. Increased sensitivity of AKT1-E17K cells to AZD5363 in the presence of HK2 inhibitor Lonidamine was reversed upon treatment with ROS inhibitor NAC. By affecting metabolism, inflammation, and redox homeostasis AKT1-E17K confers a survival advantage in meningioma cells. Our findings suggest that targeting AKT-HK2 cross-talk to induce ROS-dependent cell death could be exploited as novel therapeutic approach in meningiomas.

HKDC1 promotes tumor immune evasion in hepatocellular carcinoma by coupling cytoskeleton to STAT1 activation and PD-L1 expression.

Immune checkpoint blockade (ICB) has shown considerable promise for treating various malignancies, but only a subset of cancer patients benefit from immune checkpoint inhibitor therapy because of immune evasion and immune-related adverse events (irAEs). The mechanisms underlying how tumor cells regulate immune cell response remain largely unknown. Here we show that hexokinase domain component 1 (HKDC1) promotes tumor immune evasion in a CD8+ T cell-dependent manner by activating STAT1/PD-L1 in tumor cells. Mechanistically, HKDC1 binds to and presents cytosolic STAT1 to IFNGR1 on the plasma membrane following IFNγ-stimulation by associating with cytoskeleton protein ACTA2, resulting in STAT1 phosphorylation and nuclear translocation. HKDC1 inhibition in combination with anti-PD-1/PD-L1 enhances in vivo T cell antitumor response in liver cancer models in male mice. Clinical sample analysis indicates a correlation among HKDC1 expression, STAT1 phosphorylation, and survival in patients with hepatocellular carcinoma treated with atezolizumab (anti-PD-L1). These findings reveal a role for HKDC1 in regulating immune evasion by coupling cytoskeleton with STAT1 activation, providing a potential combination strategy to enhance antitumor immune responses.

PARP-1 inhibitor alleviates cerebral ischemia/reperfusion injury by reducing PARylation of HK-1 and LDH in mice.

Poly (ADP-ribose) polymerase-1 (PARP-1) activity significantly increases during cerebral ischemia/reperfusion. PARP-1 is an NAD+-consumption enzyme. PARP-1 hyperactivity causes intracellular NAD+ deficiency and bioenergetic collapse, contributing to neuronal death. Besides, the powerful trigger of PARP-1 causes the catalyzation of poly (ADP-ribosyl)ation (PARylation), a posttranslational modification of proteins. Here, we found that PARP-1 was activated in the ischemic brain tissue during middle-cerebral-artery occlusion and reperfusion (MCAO/R) for 24 h, and PAR accumulated in the neurons in mice. Using immunoprecipitation, Western blotting, liquid chromatography-mass spectrometry, and 3D-modeling analysis, we revealed that the activation of PARP-1 caused PARylation of hexokinase-1 and lactate dehydrogenase-B, which, therefore, caused the inhibition of these enzyme activities and the resulting cell energy metabolism collapse. PARP-1 inhibition significantly reversed the activity of hexokinase and lactate dehydrogenase, decreased infarct volume, and improved neuronal deficiency. PARP-1 inhibitor combined with pyruvate further alleviated MCAO/R-induced ischemic brain injury in mice. As such, we conclude that PARP-1 inhibitor alleviates neuronal death partly by inhibiting the PARylation of metabolic-related enzymes and reversing metabolism reprogramming during cerebral ischemia/reperfusion injury in mice. PARP-1 inhibitor combined with pyruvate might be a promising therapeutic approach against brain ischemia/reperfusion injury.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Elongation factor 1A1 regulates metabolic substrate preference in mammalian cells.

Eukaryotic elongation factor 1A1 (EEF1A1) is canonically involved in protein synthesis but also has noncanonical functions in diverse cellular processes. Previously, we identified EEF1A1 as a mediator of lipotoxicity and demonstrated that chemical inhibition of EEF1A1 activity reduced mouse liver lipid accumulation. These findings suggested a link between EEF1A1 and metabolism. Therefore, we investigated its role in regulating metabolic substrate preference. EEF1A1-deficient Chinese hamster ovary (2E2) cells displayed reduced media lactate accumulation. These effects were also observed with EEF1A1 knockdown in human hepatocyte-like HepG2 cells and in WT Chinese hamster ovary and HepG2 cells treated with selective EEF1A inhibitors, didemnin B, or plitidepsin. Extracellular flux analyses revealed decreased glycolytic ATP production and increased mitochondrial-to-glycolytic ATP production ratio in 2E2 cells, suggesting a more oxidative metabolic phenotype. Correspondingly, fatty acid oxidation was increased in 2E2 cells. Both 2E2 cells and HepG2 cells treated with didemnin B exhibited increased neutral lipid content, which may be required to support elevated oxidative metabolism. RNA-seq revealed a >90-fold downregulation of a rate-limiting glycolytic enzyme, hexokinase 2, which we confirmed through immunoblotting and enzyme activity assays. Pathway enrichment analysis identified downregulations in TNFA signaling via NFKB and MYC targets. Correspondingly, nuclear abundances of RELB and MYC were reduced in 2E2 cells. Thus, EEF1A1 deficiency may perturb glycolysis by limiting NFKB- and MYC-mediated gene expression, leading to decreased hexokinase expression and activity. This is the first evidence of a role for a translation elongation factor, EEF1A1, in regulating metabolic substrate utilization in mammalian cells.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum.

Cryptosporidium parvum is an obligate intracellular parasite with a highly reduced mitochondrion that lacks the tricarboxylic acid cycle and the ability to generate ATP, making the parasite reliant on glycolysis. Genetic ablation experiments demonstrated that neither of the two putative glucose transporters CpGT1 and CpGT2 were essential for growth. Surprisingly, hexokinase was also dispensable for parasite growth while the downstream enzyme aldolase was required, suggesting the parasite has an alternative way of obtaining phosphorylated hexose. Complementation studies in E. coli support a role for direct transport of glucose-6-phosphate from the host cell by the parasite transporters CpGT1 and CpGT2, thus bypassing a requirement for hexokinase. Additionally, the parasite obtains phosphorylated glucose from amylopectin stores that are released by the action of the essential enzyme glycogen phosphorylase. Collectively, these findings reveal that C. parvum relies on multiple pathways to obtain phosphorylated glucose both for glycolysis and to restore carbohydrate reserves.

Circ_0000376 regulates miR-577/HK2/LDHA signaling pathway to promote the growth, invasion and glycolysis of osteosarcoma.

BACKGROUND: Many studies have confirmed that circular RNAs (circRNAs) mediate the malignant progression of various tumors including osteosarcoma (OS). Our study is to uncover novel molecular mechanisms by which circ_0000376 regulates OS progression. METHODS: The expression of circ_0000376, microRNA (miR)-577, hexokinase 2 (HK2) and lactate dehydrogenase-A (LDHA) was determined by quantitative real-time PCR. OS cell proliferation, apoptosis and invasion were measured using cell counting kit 8 assay, colony formation assay, EdU assay, flow cytometry and transwell assay. Besides, cell glycolysis was assessed by testing glucose consumption, lactate production, and ATP/ADP ratios. Protein expression was examined by western blot analysis. The interaction between miR-577 and circ_0000376 or HK2/LADA was verified by dual-luciferase reporter assay. The role of circ_0000376 on OS tumor growth was explored by constructing mice xenograft models. RESULTS: Circ_0000376 had been found to be upregulated in OS tissues and cells. Functional experiments revealed that circ_0000376 interference hindered OS cell growth, invasion and glycolysis. Circ_0000376 sponged miR-577 to reduce its expression. In rescue experiments, miR-577 inhibitor abolished the regulation of circ_0000376 knockdown on OS cell functions. MiR-577 could target HK2 and LDHA in OS cells. MiR-577 suppressed OS cell growth, invasion and glycolysis, and these effects were reversed by HK2 and LDHA overexpression. Also, HK2 and LDHA expression could be regulated by circ_0000376. In vivo experiments showed that circ_0000376 knockdown inhibited OS tumorigenesis. CONCLUSION: Circ_0000376 contributed to OS growth, invasion and glycolysis depending on the regulation of miR-577/HK2/LDHA axis, providing a potential target for OS treatment.

The role of fenugreek seed extract in alleviating pancreatic toxic effects and altering glucose homeostasis induced by acetamiprid via modulation of oxidative stress, apoptosis, and autophagy.

Acetamiprid (ACMP) is a second-generation neonicotinoid that has been extensively used in the last few years. The present study examined the toxic effects of ACMP on the pancreas and glucose homeostasis through the evaluation of histological and biochemical changes and the possible ameliorative role of fenugreek seed extract (FG). Fifty adult albino rats were divided into 5 groups: negative control, positive control, FG-treated, ACMP-treated, and ACMP + FG-treated groups by oral gavage for 12 weeks. The ACMP-treated group highlighted significant elevations in plasma glucose, glycosylated haemoglobin levels (HbA1c), serum amylase, and serum lipase, along with a decrease in plasma insulin levels. In addition, significant increases in tumour necrosis factor- alpha (TNF-α) and malondialdehyde (MDA) were associated with reductions in the levels of interleukin 10 (IL-10), glutathione peroxidase, and catalase. Moreover, glucose-6-phosphatase and glycogen phosphorylase were significantly increased, with a significant reduction in hexokinase and liver glycogen stores. These biochemical changes were associated with histological changes in pancreatic sections stained by haematoxylin and eosin, Masson stain, and Orcein stain. ACMP-treated cells showed a marked reduction in ß- cell immune reactivity to insulin, with pronounced p53, and beclin 1 immune expression. The use of FG with ACMP induced partial protection except for hexokinase and glycogen phosphorylase.

DDX24 promotes tumor progression by mediating hexokinase-1 induced glycolysis in gastric cancer.

Metabolic reprogramming allows tumor cells to meet high demand of biogenesis and increased energy for rapid proliferation. Gastric cancer (GC) ranks among the most prevalent malignancies globally. Exploring the underlying mechanisms of glycolytic reprogramming in GC could provide new therapeutic target for GC treatment. Here, we showed that DEAD-box helicase 24 (DDX24) played a critical role in hexokinase-1 (HK1) induced glycolysis. DDX24 expression was significantly elevated in GC tissues and was closely associated with worse survival in GC patients. In addition, DDX24 promoted glucose uptake and lactate production in GC cells. Mechanistically, DDX24 could bind the HK1 mRNA and positively regulated HK1 level at the transcriptional level. Moreover, DDX24 promoted the proliferation, migration, and invasion ability of GC cells by upregulating HK1. Collectively, these results suggested that DDX24 was a critical player in the regulation of glycolytic reprogramming and also implicated DDX24 as a valuable therapeutic target for GC.