Neuron-specific deficits of bioenergetic processes in the dorsolateral prefrontal cortex in schizophrenia.

Schizophrenia is a devastating illness that affects over 2 million people in the United States and costs society billions of dollars annually. New insights into the pathophysiology of schizophrenia are needed to provide the conceptual framework to facilitate development of new treatment strategies. We examined bioenergetic pathways in the dorsolateral prefrontal cortex (DLPFC) of subjects with schizophrenia and control subjects using western blot analysis, quantitative real-time polymerase chain reaction, and enzyme/substrate assays. Laser-capture microdissection-quantitative polymerase chain reaction was used to examine these pathways at the cellular level. We found decreases in hexokinase (HXK) and phosphofructokinase (PFK) activity in the DLPFC, as well as decreased PFK1 mRNA expression. In pyramidal neurons, we found an increase in monocarboxylate transporter 1 mRNA expression, and decreases in HXK1, PFK1, glucose transporter 1 (GLUT1), and GLUT3 mRNA expression. These results suggest abnormal bioenergetic function, as well as a neuron-specific defect in glucose utilization, in the DLPFC in schizophrenia.

Enhanced Diffusion and Oligomeric Enzyme Dissociation.

The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but experimental support has involved oligomeric enzymes, often studied under conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds kM, the Michaelis constant. Urease, a hexamer of subunits, has been considered to be the gold standard demonstrating enhanced diffusion. Here we show that urease and certain other oligomeric enzymes dissociate above kM into their subunits that diffuse more rapidly, thus providing a simple physical mechanism that contributes to enhanced diffusion in this regime of concentrations. Mindful that this conclusion may be controversial, our findings are supported by four independent analytical techniques: static light scattering, dynamic light scattering (DLS), size-exclusion chromatography (SEC), and fluorescence correlation spectroscopy (FCS). Data for urease are emphasized and the conclusion is validated for hexokinase, acetylcholinesterase, and aldolase. For hexokinase and aldolase no enhanced diffusion is observed except under conditions when these oligomeric enzymes dissociate. At substrate concentration regimes below kM at which acetylcholinesterase and urease do not dissociate, our finding showing up to 10% enhancement of the diffusion coefficient is consistent with various theoretical scenarios in the literature.

cAMP Receptor Protein of Salmonella enterica Serovar Typhimurium Modulate Glycolysis in Macrophages to Induce Cell Apoptosis.

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Hexokinase 2 drives glycogen accumulation in equine endometrium at day 12 of diestrus and pregnancy.

BACKGROUND: Secretion of histotroph during the prolonged pre-implantation phase in mares is crucial to pregnancy maintenance, manifested as increased embryonic loss in mares with age-related endometrial degeneration. Glycogen content of uterine histotroph is higher during the progesterone-dominated phase of the estrous cycle in mares, but regulatory mechanisms are not well understood. METHODS: mRNA expression of glycogen-metabolizing enzymes (HK1, HK2, GSK3B, GYS1, PEPCK, PKM, PYGM) in endometrial samples were compared among mares in anestrus, estrus, and at Day 12 of diestrus and pregnancy. In addition, hexokinase 2 (HK2) activity was assessed using a colorimetric assay. RESULTS: HK2 was the key regulator of glycogen accumulation during diestrus and pregnancy; hexokinase transcript abundance and enzyme activity were significantly higher during diestrus and pregnancy than estrus and anestrus. In addition, despite similar relative transcript abundance, hexokinase activity was significantly greater in the pregnant versus diestrous endometrium. Therefore, we inferred there was regulation of hexokinase activity through phosphorylation, in addition to its regulation at the transcriptional level during early pregnancy. Based on immunohistochemistry, HK2 was localized primarily in luminal and glandular epithelial cells, with weaker staining in stromal cells. CONCLUSION: Among glycogen metabolizing enzymes identified, expression of HK2 was significantly greater during the progesterone-dominated phase of the cycle.

Molecular Cloning and Functional Characterization of a Hexokinase from the Oriental River Prawn Macrobrachium nipponense in Response to Hypoxia.

Metabolic adjustment to hypoxia in Macrobrachium nipponense (oriental river prawn) implies a shift to anaerobic metabolism. Hexokinase (HK) is a key glycolytic enzyme in prawns. The involvement of HK in the hypoxia inducible factors (HIFs) pathway is unclear in prawns. In this study, the full-length cDNA for HK (MnHK) was obtained from M. nipponense, and its properties were characterized. The full-length cDNA (2385 bp) with an open reading frame of 1350 bp, encoded a 450-amino acid protein. MnHK contained highly conserved amino acids in the glucose, glucose-6-phosphate, ATP, and Mg+2 binding sites. Quantitative real-time reverse transcription PCR assays revealed the tissue-specific expression pattern of MnHK, with abundant expression in the muscle, and gills. Kinetic studies validated the hexokinase activity of recombinant HK. Silencing of HIF-1α or HIF-1ß subunit genes blocked the induction of HK and its enzyme activities during hypoxia in muscles. The results suggested that MnHK is a key factor that increases the anaerobic rate, and is probably involved in the HIF-1 pathway related to highly active metabolism during hypoxia.

Evidence for heightened hexokinase II immunoexpression in hepatocyte dysplasia and hepatocellular carcinoma.

BACKGROUND: Normal hepatocytes exhibit low-affinity hexokinase (glucokinase [HKIV]), but during oncogenesis, there is a switch from HKIV to HKII expression. The aims of this study were to compare the immunoexpression of HKII in non-dysplastic cirrhosis (NDC), liver cell change/dysplasia in cirrhosis (LCD), HCC, and normal liver control tissues, and to correlate HKII expression with clinical and histopathological parameters. DESIGN: Immunohistochemistry was performed on a liver cancer progression tissue array consisting of specimens from explants with cirrhosis, including 45 tissue samples with HCC, 108 without HCC, 143 with LCD, and 8 normal liver control tissues. HKII expression was quantified as positive pixel counts/square millimeter (ppc/mm(2)) by image analysis. RESULTS: There was a stepwise increase in HKII level from normal liver tissue to NDC, to LCD, and to HCC (p = 0.001). HKII levels were significantly higher in areas of LCD versus NDC (p ≤ 0.001), and in LCD and HCC versus NDC (p = 0.007). HKII levels were similar in LCD and HCC (p = 0.124). HKII levels were higher in grade 2-4 versus grade 1 HCCs (p = 0.044), and in pleomorphic versus non-pleomorphic HCC variants (p = 0.041). Higher levels of HKII expression in LCD and HCC versus NDC and in higher tumor grade remained significant in multivariate analysis. CONCLUSIONS: Higher levels of HKII immunoexpression in LDC and HCC compared with NDC suggest that upregulation of HKII occurs during the process of hepatocarcinogenesis in humans. In HCC, higher levels of HKII are associated with more aggressive histological features.

Hexokinase 2 overexpression promotes the proliferation and survival of laryngeal squamous cell carcinoma.

Proliferating cancer cells preferentially use anaerobic glycolysis rather than oxidative phosphorylation for energy production. Hexokinase 2 (HK2) is highly expressed in many malignant cells and is necessary for anaerobic glycolysis. The role of HK2 in laryngeal squamous cell carcinoma (LSCC) is unknown. In this study, the expression of HK2 in LSCC was investigated and the effect of inhibiting HK2 expression with small hairpin RNA (shRNA) on tumor growth was investigated. Using immunohistochemistry, HK2 expression was assessed in LSCC tissues. Human laryngeal carcinoma Hep-2 cells were stably transfected with a plasmid expressing HK2 shRNA (pGenesil-1.1-HK2) and were compared to control cells with respect to the cell cycle, cell viability, apoptosis, and their ability to form xenograft tumors. HK2 expression was significantly higher in LSCC than in papilloma or glottis polypus. Tumor samples of higher T, N, and TNM stage often had stronger HK2 staining. HK2 shRNA reduced HK2 mRNA, protein levels, and HK activity in Hep-2 cells. HK2 cells expressing shRNA demonstrated a higher G0-G1 ratio, increased apoptosis, and reduced viability. Xenograft tumors derived from cells expressing HK2 shRNA were smaller and had lower proliferation than those from untransfected or control-plasmid-transfected cells. In conclusion, depletion of HK2 expression resulted in reduced xenograft tumor development likely by reducing proliferation, altering the cell cycle, reducing cell viability and activating apoptosis. These data suggest that HK2 plays an important role in the development of LSCC and represents a potential therapeutic target for LSCC.

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.

Expression of hexokinase 2 in epithelial ovarian tumors and its clinical significance in serous ovarian cancer.

"Warburg effect" emphasizes that malignant cells exhibit active glycolysis even under aerobic conditions. Hexokinase 2 (HK2) is a key glycolytic enzyme that helps to exhibit a "Warburg effect". In the present study, the main aim was to detect the expression of HK2 in epithelial ovarian tumor tissues. Immunohistochemistry and qRT-PCR were used to examine the expression of HK2 in different epithelial ovarian tissues. The expression of HK2 in ovarian cancer tissues was significantly higher than that in normal ovarian, benign, and borderline tumors both in protein (p < 0.001) and mRNA (p < 0.05) levels. HK2 expression was significantly higher in Stage III/IV compared to Stage III (p < 0.001). Expression of HK2 in poorly-differentiated carcinoma was higher than that in well-differentiated carcinoma (p = 0.008). The level of HK2 was higher in serous groups than in non-serous groups in both protein (p = 0.008) and mRNA (p < 0.05) level. Collectively, HK2 is highly expressed in epithelial ovarian cancer, especially in serous groups. Its expression is related with clinical stage and histological differentiation.

Hexokinase II in breast carcinoma: a potent prognostic factor associated with hypoxia-inducible factor-1α and Ki-67.

Hypoxia-inducible factor-1α (HIF-1α) mediates adaptive responses to changes under tissue hypoxia in carcinoma cells by controlling the expression of various target genes. Previous studies have demonstrated that HIF-1α is associated with adverse clinical outcome in breast carcinoma patients, but details of HIF-1α's role have remained largely unknown. Therefore, in this study, we examined the expression profiles of HIF-1α-induced genes in 10 breast carcinoma cases using microarray data. As a result, we demonstrated that the status of hexokinase II (HKII) was associated with carcinoma recurrence in patients with these genes. The enzyme HKII is involved in the first, and rate-limiting, step of glycolysis, but its clinical significance has not yet been examined in breast carcinoma. Therefore, we immunolocalized HKII in 118 breast carcinomas, and HKII immunoreactivity was detected in 44% of the cases. It is significantly associated with histological grade, Ki-67 labeling index and HIF-1α immunoreactivity. Also, HKII status is significantly associated with increased risk of recurrence and adverse clinical outcome in breast cancer patients. Subsequent multivariate analysis demonstrated that HKII status was an independent prognostic factor for disease-free survival of patients. These results all suggest that HKII is induced by HIF-1α and plays important roles in the proliferation and/or progression of breast carcinoma possibly through increased glycolytic activity. The status of HKII is therefore considered a potent prognostic factor in human breast cancer patients.

Right-to-left ventricular differences in the expression of mitochondrial hexokinase and phosphorylation of Akt.

BACKGROUND/AIMS: Hexokinase (HK) is a key glycolytic enzyme which promotes the maintenance of glucose homeostasis in cardiomyocytes. HK1 isoform is predominantly bound to the outer mitochondrial membrane and highly supports oxidative phosphorylation by increasing the availability of ADP for complex V of the respiratory chain. HK2 isoform is under physiological conditions predominantly localized in the cytosol and upon stimulation of PI3K/ Akt pathway associates with mitochondria and thus can prevent apoptosis. The purpose of this study was to investigate expression and subcellular localization of both HK isoforms in left (LV) and right (RV) heart ventricles of adult male Wistar rats. METHODS: Real-Time RT-PCR, Western blotting, and quantitative immunofluorescence microscopy were used. RESULTS: Our results showed a significantly higher expression of both HK1 and HK2 at mRNA and protein levels in the RV compared to the LV. These findings were corroborated by immunofluorescence staining which revealed substantially higher fluorescence signals of both HKs in the RV than in the LV. The ratios of phospho-Ser473-Akt/non-phospho-Akt and phospho-Thr308-Akt/non-phospho-Akt were also markedly higher in the RV than in the LV. CONCLUSION: These results suggest that the RV has a higher activity of aerobic glycolytic metabolism and may be able to respond faster and more powerfully to stressful stimuli than the LV.

Visual detection of hexokinase activity and inhibition with positively charged gold nanoparticles as colorimetric probes.

Positively charged gold nanoparticles can effectively differentiate ATP and ADP, thus providing a simple and visual approach to colorimetric detection of hexokinase activity and inhibition.

Hexokinase 2 promoted cell motility and proliferation by activating Akt1/p-Akt1 in human ovarian cancer cells.

BACKGROUND: Recently, increasing evidence has indicated that elevation of Hexokinase 2 (HK2) plays an important role in several cancers on regulating cell motility and growth. However, its role on regulating cell EMT in human ovarian cancer still less to known. METHODS: The transwell and wound-healing assay were used to detect the effective of HK2 on regulating motility of ovarian cancer cells. Real Time PCR and Western Blotting were used to explore the changing of EMT-related proteins in HK2-modified cells. The clonogenic formation, cell growth curves and MTT assays were used to evaluate the effective of HK2 on regulating cell proliferation in HK2-modified cells. The flow cytometry was used to detect the differences in the distribution of cells in the cell cycle between the HK2-modified cells and their control cells. The correlation of HK2 and Akt1/p-Akt1 was explored by using Western Blotting, Akt1 inhibitor (MK2206) and transient transfection of an Akt1 recombinant plasmid. The potential correlation between HK2 and EMT-related proteins in human ovarian cancer tissues and OV (ovarian serous cystadenocarcinoma) was confirmed by using Pearson correlation analysis and TIMER 2.0. RESULTS: In ovarian cancer cells, overexpressing of HK2 enhanced cell motility by inducing of EMT-related proteins, such as CDH2, fibronectin, MMP9, ZEB1, ZEB2 and vimentin. Moreover, overexpressing of HK2 promoted cell growth by reducing p21 and p27 expression in ovarian cancer cells. Further studies demonstrated that this promotion of cell motility and growth by HK2 was probably a result of it activating of Akt1 (p-Akt1) in ovarian cancer cells. Additionally, the positive correlation between HK2 and p-Akt1, fibronectin, MMP9 expression in human ovarian cancer samples was verified by using Pearson correlation analysis. The positive correlation between HK2 and CDH2, fibronectin, MMP9, ZEB1, ZEB2 and vimentin in OV (ovarian serous cystadenocarcinoma) was confirmed by using TIMER 2.0. CONCLUSION: This study demonstrated that HK2 could induce EMT-related proteins and reduce cell cycle inhibitor by activating Akt1 in human ovarian cancer cells, subsequently enhancing cell motility and growth, suggesting that HK2 participate in the malignant process of ovarian cancer by interacting with Akt1.

Increased hexokinase-2 as a novel biomarker for the diagnosis and correlating with disease severity in rheumatoid arthritis.

ABSTRACT: Abnormal glucose metabolism brings out joint inflammation and destruction in rheumatoid arthritis (RA). The aim of this study was to evaluate the potential of circulating hexokinase-2 (HK2) in peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis (RA) patients.PBMCs were obtained from patients with RA or osteoarthritis (OA) and healthy controls (HCs). The expression of HK2 was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The C-reactive protein (CRP) level, erythrocyte sedimentation rate (ESR), Calprotectin, rheumatoid factor (RF), anti-cyclic citrullinated peptides (anti-CCP) antibody level and 28-joint Disease Activity Score (DAS28), Clinical Disease Activity Index (CDAI) and Simplified Disease Activity Index (SDAI) were measured. Spearman's analysis was performed to determine the association between the level of HK2 and clinical characteristics. A receiver operating characteristic (ROC) curve was employed to evaluate the diagnostic value of HK2 in PBMCs. Logistic regression was used to identify risk factors. Sixty-five RA patients, 35 OA patients, and 40 HCs were included in the study.HK2 was upregulated in RA and OA patients compared with that in HCs (P < .05). The area under the ROC of HK2 for diagnosing RA and OA was 0.808 and 0.640, respectively. In addition, HK2 levels were increased in active RA compared with those in remittent RA (P = .03). Furthermore, HK2 correlated positively with the DAS28-ESR (P < .001), CDAI (P = .02) and SDAI scores (P = .02). Moreover, HK2 was independently associated with an increased risk of disease activity (DAS28-ESR>3.2, P = .02; CDAI score>10, P = .03; SDAI score>11, P = .04). Additionally, HK2 positivity was more frequently detected in patients treated with biologic disease-modifying antirheumatic drugs (bDMARDs) than in those not treated with bDMARDs.HK2 levels in PBMCs can be considered an ideal biomarker for diagnosing RA and involved in disease activity in RA. Dysregulation of HK2 may participate in the molecular mechanism of RA and could be an attractive selective metabolic target for RA treatment.

Kinetics of transport and phosphorylation of glucose in cancer cells.

Metabolic control analysis of tumor glycolysis has indicated that hexokinase (HK) and glucose transporter (GLUT) exert the main flux control (71%). To understand why they are the main controlling steps, the GLUT and HK kinetics and the contents of GLUT1, GLUT2, GLUT3, GLUT4, HKI, and HKII were analyzed in rat hepatocarcinoma AS-30D and HeLa human cervix cancer. An improved protocol to determine the kinetic parameters of GLUT was developed with D-[2-(3)H-glucose] as physiological substrate. Kinetic analysis revealed two components at low- and high-glucose concentrations in both tumor cells. At low glucose and 37 degrees C, the V(max) was 55 +/- 20 and 17.2 +/- 6 nmol (min x mg protein)(-1), whereas the K(m) was 0.52 +/- 0.7 and 9.3 +/- 3 mM for hepatoma and HeLa cells, respectively. GLUT activity was partially inhibited by cytochalasin B (IC(50) = 0.44 +/- 0.1; K(i) = 0.3 +/- 0.1 microM) and phloretin (IC(50) = 8.7 microM) in AS-30D hepatocarcinoma. At physiological glucose, GLUT1 and GLUT3 were the predominant active isoforms in HeLa cells and AS-30D cells, respectively. HK activity in HeLa cells was much lower (60 mU/mg protein) than that in AS-30D cells (700 mU/mg protein), but both HKs were strongly inhibited by G6P. HKII was the predominant isoform in AS-30D carcinoma and HeLa cells. The much lower GLUT V(max) and catalytic efficiency (V(max)/K(m)) values in comparison to those of G6P-sensitive HK suggested the transporter exerts higher control on the glycolytic flux than HK in cancer cells. Thus, GLUT seems a more adequate therapeutic target.

Modulation of hexokinase association with mitochondria analyzed with quantitative three-dimensional confocal microscopy.

Hexokinase isozyme I is proposed to be associated with mitochondria in vivo. Moreover, it has been suggested that this association is modulated in coordination with changes in cell metabolic state. To test these hypotheses, we analyzed the subcellular distribution of hexokinase relative to mitochondria in paraformaldehyde-fixed astrocytes using immunocytochemistry and quantitative three-dimensional confocal microscopy. Analysis of the extent of colocalization between hexokinase and mitochondria revealed that approximately 70% of cellular hexokinase is associated with mitochondria under basal metabolic conditions. In contrast to the immunocytochemical studies, between 15 to 40% of cellular hexokinase was found to be associated with mitochondria after fractionation of astrocyte cultures depending on the exact fractionation conditions. The discrepancy between fractionation studies and those based on imaging of distributions in fixed cells indicates the usefulness of using techniques that can evaluate the distributions of "cytosolic" enzymes in cells whose subcellular ultrastructure is not severely disrupted. To determine if hexokinase distribution is modulated in concert with changes in cell metabolism, the localization of hexokinase with mitochondria was evaluated after inhibition of glucose metabolism with 2-deoxyglucose. After incubation with 2-deoxyglucose there was an approximate 35% decrease in the amount of hexokinase associated with mitochondria. These findings support the hypothesis that hexokinase is bound to mitochondria in rat brain astrocytes in vivo, and that this association is sensitive to cell metabolic state.

Hexokinase isoenzymes in liver and adipose tissue of man and dog.

A hexokinase, with a low Michaelis constant, not previously described, has been found in extracts of human and dog liver but not of rat liver. Earlier reports are contradicted in that glucokinase occurs in extracts of liver from well-nourished humans and dogs; it is absent, or almost so, during states of poor nutrition.

Testis-specific and sex-associated hexokinases in Drosophila melanogaster.

Hexokinases of Drosophila melanogaster were investigated by starchgel electrophoresis. A hexokinase is present in both sexes during earlier stages of development, but it persists only in male adults. In addition, in this species there is a testis-specific hexokinase which is first observed during the pupal period.

Hexokinase isoenzymes in human erythrocytes: association of type II with fetal hemoglobin.

Hexokinase activity in human erythrocytes is associated with three electrophoretically distinct bands. Normal adult erythrocytes contain hexokinases Types I and III. Type II hexokinase is present in the erythrocytes of newborn infants and absent from those of normal adults; it is, however, present in erythrocytes of adults with hereditary persistence of fetal hemoglobin. Type II hexokinase and fetal hemtoglobin appear to be associated.

Superresolution three-dimensional images of fluorescence in cells with minimal light exposure.

Fluorescent probes offer insight into the highly localized and rapid molecular events that underlie cell function. However, methods are required that can efficiently transform the limited signals from such probes into high-resolution images. An algorithm has now been developed that produces highly accurate images of fluorescent probe distribution inside cells with minimal light exposure and a conventional light microscope. This method provides resolution nearly four times greater than that currently available from any fluorescence microscope and was used to study several biological problems.