Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2)

Impaired macroautophagy/autophagy and high levels of glycolysis are prevalent in liver cancer. However, it remains unknown whether there is a regulatory relationship between autophagy and glycolytic metabolism. In this study, by utilizing cancer cells with basal or impaired autophagic flux, we demonstrated that glycolytic activity is negatively correlated with autophagy level. The autophagic degradation of HK2 (hexokinase 2), a crucial glycolytic enzyme catalyzing the conversion of glucose to glucose-6-phosphate, was found to be involved in the regulation of glycolysis by autophagy. The Lys63-linked ubiquitination of HK2 catalyzed by the E3 ligase TRAF6 was critical for the subsequent recognition of HK2 by the autophagy receptor protein SQSTM1/p62 for the process of selective autophagic degradation. In a tissue microarray of human liver cancer, the combination of high HK2 expression and high SQSTM1 expression was shown to have biological and prognostic significance. Furthermore, 3-BrPA, a pyruvate analog targeting HK2, significantly decreased the growth of autophagy-impaired tumors in vitro and in vivo (p < 0.05). By demonstrating the regulation of glycolysis by autophagy through the TRAF6- and SQSTM1-mediated ubiquitination system, our study may open an avenue for developing a glycolysis-targeting therapeutic intervention for treatment of autophagy-impaired liver cancer.     

Retinal Hexokinase: Kinetic Properties and the Effect of Cyclic 3',5'-Adenosine Monophosphate

Abstract: We measured hexokinase (EC 2.7.1.1) activity in particulate and soluble fractions isolated from bullfrog ( Rana catesbeiana ) retinas. Seventy-three percent of the hexokinase (HK) activity was associated with the particulate fraction, 27% with the soluble fraction. Both HK fractions could phosphorylate fructose, glucose, 2-deoxy-d-glucose, and mannose, but not galactose. The K _m for glucose was 0.14 m M , for 2-deoxy-d-glucose. 3.6 m M . With glucose as substrate, the V _max for particulate HK was 125–148 μ M retina⁻¹ min⁻¹, for soluble HK, 37 μ M retina⁻¹ min⁻¹. Product inhibition of particulate HK activity by glucose 6-phosphate was marked, whereas 2-deoxy-d-glucose 6-phosphate did not inhibit the activity. Cyclic AMP stimulated the HK activity of both retinal fractions nearly twofold at concentrations of 0.2–0.8 m M; AMP was much less effective in this regard.     

MicroRNA-216b targets HK2 to potentiate autophagy and apoptosis of breast cancer cells via the mTOR signaling pathway

Patients suffering from breast cancer (BC) still have a poor response to treatments, even though early detection and improved therapy have contributed to a reduced mortality. Recent studies have been inspired on the association between microRNAs (miRs) and therapies of BC. The current study set out to investigate the role of miR-216b in BC, and further analyze the underlining mechanism. Firstly, hexokinase 2 (HK2) and miR-216b were characterized in BC tissues and cells by RT-qPCR and Western blot assay. In addition, the interaction between HK2 and miR-216b was analyzed using dual luciferase reporter assay. BC cells were further transfected with a series of miR-216b mimic or inhibitor, or siRNA targeting HK2, so as to analyze the regulatory mechanism of miR-216b, HK2 and mammalian target of rapamycin (mTOR) signaling pathway, and to further explore their regulation in BC cellular behaviors. The results demonstrated that HK2 was highly expressed and miR-216b was poorly expressed in BC cells and tissues. HK2 was also verified as a target of miR-216b with online databases and dual luciferase reporter assay. Functionally, miR-216b was found to be closely associated with BC progression via inactivating mTOR signaling pathway by targeting HK2. Moreover, cell viability, migration and invasion were reduced as a result of miR-216b upregulation or HK2 silencing, while autophagy, cell cycle arrest and apoptosis were induced. Taken together, our findings indicated that miR-216b down-regulates HK2 to inactivate the mTOR signaling pathway, thus inhibiting the progression of BC. Hence, this study highlighted a novel target for BC treatment.     

HK2/hexokinase-II integrates glycolysis and autophagy to confer cellular protection

Hexokinases (HKs) catalyze the first step of glucose metabolism, phosphorylating glucose to glucose 6-phosphate (G6P). HK2/hexokinase-II is a predominant isoform in insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissues and is also upregulated in many types of tumors associated with enhanced aerobic glycolysis (the Warburg effect). Accumulating evidence indicates that HK2 plays an important role not only in glycolysis but also in cell survival. Although there is increasing recognition that cellular metabolism and cell survival are closely related, the molecular link between metabolism and autophagic pathways has not been fully elucidated. We recently discovered that HK2 facilitates autophagy in response to glucose deprivation (HK substrate deprivation) to protect cardiomyocytes, and suggest that HK2 functions as a molecular switch from glycolysis to autophagy to ensure cellular energy homeostasis under starvation conditions.     

A reevaluation of the roles of hexokinase I and II in the heart

Hexokinase is responsible for glucose phosphorylation, a process fundamental to regulating glucose uptake. In some tissues, hexokinase translocates to the mitochondria, thereby increasing its efficiency and decreasing its susceptibility to product inhibition. It may also decrease free radical formation in the mitochondria and prevent apoptosis. Whether hexokinase translocation occurs in the heart is controversial; here, using immunogold labeling for the first time, we provide evidence for this process. Rat hearts (6 groups, n = 6/group), perfused with either glucose- or glucose + oleate (0.4 mmol/l)-containing buffer, were exposed to 30-min insulin stimulation, ischemia, or control perfusion. Hexokinase I (HK I) and hexokinase II (HK II) distributions were then determined. In glucose-perfused hearts, HK I-mitochondrial binding increased from 0.41 +/- 0.04 golds/mm in control hearts to 0.71 +/- 0.10 golds/mm after insulin and to 1.54 +/- 0.38 golds/mm after ischemia (P < 0.05). Similarly, HK II-mitochondrial binding increased from 0.16 +/- 0.02 to 0.53 +/- 0.08 golds/mm with insulin and 0.44 +/- 0.07 golds/mm after ischemia (P < 0.05). Under basal conditions, the fraction of HK I that was mitochondrial bound was five times greater than for HK II; insulin and ischemia caused a fourfold increase in HK II binding but only a doubling in HK I binding. Oleate decreased hexokinase-mitochondrial binding and abolished insulin-mediated translocation of HK I. Our data show that mitochondrial-hexokinase binding increases under insulin or ischemic stimulation and that this translocation is modified by oleate. These events are isoform specific, suggesting that HK I and HK II are independently regulated and implying that they perform different roles in cardiac glucose regulation.     

Rabbit heart mitochondrial hexokinase: Solubilization and general properties

In rabbit heart, results show that two isoenzymes of hexokinase (HK) are present. The enzymatic activity associated with mitochondria consists of only one isoenzyme; according to its electrophoretic mobility and its apparent K_m for glucose (0.065 mm), it has been identified as type I isoenzyme. The bound HK I exhibits a lower apparent K_m for ATPMg than the solubilized enzyme, whereas the apparent K_m for glucose is the same for bound and solubilized HK. Detailed studies have been performed to investigate the interactions which take place between the enzyme and the mitochondrial membrane. Neutral salts efficiently solubilize the bound enzyme. Digitonin induces only a partial release of the enzyme bound to mitochondria; this result could be explained by the existence of contacts between the outer and the inner mitochondrial membranes [C. R. Hackenbrock (1968)Proc. Natl. Acad. Sci. USA61, 598–605]. Furthermore, low concentrations (0.1 mm) of glucose 6-phosphate (G6P) or ATP^4? specifically solubilize hexokinase. The solubilizing effect of G6P and ATP^4?, which are potent inhibitors of the enzyme, can be prevented by incubation of mitochondria with P_i or Mg²⁺. In addition, enzyme solubilization by G6P can be reversed by Mg²⁺ only when the proteolytic treatment of the heart homogenate is omitted during the course of the isolation of mitochondria. These results concerning the interaction of rabbit heart hexokinase with the outer mitochondrial membrane agree with the schematic model proposed by Wilson [(1982) Biophys. J.37, 18–19] for the brain enzyme. This model involves the existence of two kinds of interactions between HK and mitochondria; a very specific one with the hexokinase-binding protein of the outer mitochondrial membrane, which is suppressed by glucose 6-phosphate, and a less specific, cation-mediated one.     

VDAC1 serves as a mitochondrial binding site for hexokinase in oxidative muscles

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are the main pathway for metabolites across the mitochondrial outer membrane and may serve as binding sites for kinases, including hexokinase. We determined that mitochondria-bound hexokinase activity is significantly reduced in oxidative muscles (heart and soleus) in vdac1^−/− mice. The activity data were supported by western blot analysis using HK2 specific antibody. To gain more insight into the physiologic mean of the results with the activity data, VDAC deficient mice were subjected to glucose tolerance testing and exercise-induced stress, each of which involves tissue glucose uptake via different mechanisms. vdac1^−/− mice exhibit impaired glucose tolerance whereas vdac3^−/− mice have normal glucose tolerance and exercise capacity. Mice lacking both VDAC1 and VDAC3 (vdac1^−/−/vdac3^−/−) have reduced exercise capacity together with impaired glucose tolerance. Therefore, we demonstrated a link between VDAC1 mediated mitochondria-bound hexokinase activity and the capacity for glucose clearance.     

Degradation of HK2 by chaperone-mediated autophagy promotes metabolic catastrophe and cell death

Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non–acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.     

Change in cell membrane permeability, and composition and properties of hexokinase during induced carcinogenesis]

The isoenzyme hexokinase (HK) spectrum from normal rat large intestinal mucosa consisted of 3 isoenzymes. In tumours of this localization induced by 1,2-dimethylhydrazine there proved to be a lack or marked decrease in the most rapid anodic isoenzyme. Only one HK isoenzyme was found in the metastases. Km (glucose) for tumour HK was 2--3 times lower than for normal intestinal HK; the HK activity was detected in the serum from the 1st month of the carcinogenic administration, and by the 5th month it was found in 80% of the tumour-bearing animals. No serum HK activity was ever found in control rats.     

Hexokinase II partial knockout impairs exercise-stimulated glucose uptake in oxidative muscles of mice

Muscle glucose uptake (MGU) is distributively controlled by three serial steps: delivery of glucose to the muscle membrane, transport across the muscle membrane, and intracellular phosphorylation to glucose 6-phosphate by hexokinase (HK). During states of high glucose fluxes such as moderate exercise, the HK activity is of increased importance, since augmented muscle perfusion increases glucose delivery, and increased GLUT4 at the cell membrane increases glucose transport. Because HK II overexpression augments exercise-stimulated MGU, it was hypothesized that a reduction in HK II activity would impair exercise-stimulated MGU and that the magnitude of this impairment would be greatest in tissues with the largest glucose requirement. To this end, mice with a HK II partial knockout (HK+/-) were compared with their wild-type control (WT) littermates during either sedentary or moderate exercise periods. Rg, an index of glucose metabolism, was measured using 2-deoxy-[3H]glucose. No differences in glucose metabolism were detected between sedentary groups. The increase in Rg due to exercise was impaired in the highly oxidative heart and soleus muscles of HK+/- compared with WT mice (7 +/- 10 vs. 29 +/- 9 and 8 +/- 3 vs. 25 +/- 7 micromol. 100 g-1. min-1, respectively). However, the increase in Rg due to exercise was not altered in gastrocnemius and superficial vastus lateralis muscles in HK+/- and WT mice (8 +/- 2 vs. 12 +/- 3 and 5 +/- 2 vs. 8 +/- 2 micromol. 100 g-1. min-1, respectively). In conclusion, MGU is impaired by reductions in HK activity during exercise, a physiological condition characterized by high glucose flux. This impairment is critically dependent on the tissue's glucose metabolic rate and correlates with tissue oxidative capacity.     

Mitochondrial hexokinase from differentiated and undifferentiated HT29 colon cancer cells: effect of some metabolites on the bound/soluble equilibrium

1. Solubilization of mitochondrial bound hexokinase (HK), which represents 75-80% of the total enzyme activity in the cells, was investigated in freshly isolated mitochondria from undifferentiated (Glc+) or differentiated (Glc-) HT29 adenocarcinoma cells. In both models, the bound HK is almost completely released in vitro by 100 microM glucose 6-P (G 6-P). 2. Free ATP (5 mM) or palmitate (800 microM) produce a partial solubilization of bound HK, more markedly in the case of Glc- mitochondria. 3. Glucose or glucose 1-P are found unable to solubilize bound HK. Glucose 1,6-P2, 2-deoxyglucose 6-P or glucosamine 6-P can solubilize the enzyme but are less efficient than G 6-P. 4. Mg2+ and Pi are found to counteract the glucose 6-P induced solubilization of HK in both types of mitochondria. Taking into account the intracellular concentrations of these ions, this could in part explain why, in HT29 cells, HK is predominantly bound to the mitochondria.     

An electrophoretic study of the distribution and properties of human hexokinases

An electrophoretic system which gives a clear separation of human hexokinases HK1, HK2 and HK3 is described. The distribution of the hexokinase isozymes in various human tissues, both adult and fetal, is reported. Some properties of the isozymes were investigated. HK2 was found to be more thermolabile than HK1, and there was also a small but significant difference in molecular size. Unlike HK3, HK1 and HK2 are not inhibited by high glucose concentrations. Screening of red cell lysates from 800 unrelated European individuals revealed no genetic variants of HK1 and HK2. However, in view of their difference in properties, it seems probable that the HK1 and HK2 isozymes are determined by separate gene loci.     

Cloning, expression, and characterization of the hxk-1 Gene from the white truffle Tuber borchii vittad.: A first step toward understanding sugar metabolism.

Recent biochemical investigations of Tuber borchii Vittad. mycelium have demonstrated the presence of three distinct forms of hexokinase (HK(M1), HK(M2), and HKM3). In the investigation described here, a gene coding for hexokinase (hxk-1) from T. borchii was isolated and characterized. The hxk-1 gene is characterized by an ORF of 1494 nucleotides and codes for a polypeptide of 497 aa. The gene was overexpressed in Escherichia coli, and the recombinant protein was kinetically characterized. The K(cat) value for fructose is in agreement with the data reported for the hexokinase of Yarrowia lipolytica, the Km for ATP is not dependent on the sugar used, and the enzyme is not inhibited by trehalose 6-phosphate or glucose 6-phosphate. The biochemical characteristics confirm that this enzyme is a hexokinase, as suggested by the Pileup results, and it corresponds to the HKM1 isoform. This work represents the first characterization of the key enzyme of the glycolytic pathway and the related gene in a Tuber species.     

The Futile Cycling of Hexose Phosphates Could Account for the Fact That Hexokinase Exerts a High Control on Glucose Phosphorylation but Not on Glycolytic Rate in Transgenic Potato (Solanum tuberosum) Roots

The metabolism of potato (Solanum tuberosum) roots constitutively over- and underexpressing hexokinase (HK, EC 2.7.1.1) was examined. An 11-fold variation in HK activity resulted in altered root growth, with antisense roots growing better than sense roots. Quantification of sugars, organic acids and amino acids in transgenic roots demonstrated that the manipulation of HK activity had very little effect on the intracellular pools of these metabolites. However, adenylate and free Pi levels were negatively affected by an increase in HK activity. The flux control coefficient of HK over the phosphorylation of glucose was measured for the first time in plants. Its value varied with HK level. It reached 1.71 at or below normal HK activity value and was much lower (0.32) at very high HK levels. Measurements of glycolytic flux and O₂ uptake rates demonstrated that the differences in glucose phosphorylation did not affect significantly glycolytic and respiratory metabolism. We hypothesized that these results could be explained by the existence of a futile cycle between the pools of hexose-Ps and carbohydrates. This view is supported by several lines of evidence. Firstly, activities of enzymes capable of catalyzing these reactions were detected in roots, including a hexose-P phosphatase. Secondly, metabolic tracer experiments using ¹⁴C-glucose as precursor showed the formation of ¹⁴C-fructose and ¹⁴C-sucrose. We conclude that futile cycling of hexose-P could be partially responsible for the differences in energetic status in roots with high and low HK activity and possibly cause the observed alterations in growth in transgenic roots. The involvement of HK and futile cycles in the control of glucose-6P metabolism is discussed.     

Computational prediction and experimental validation of the activator function of C2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone on pancreatic and hepatic hexokinase

Abstract

This study identifies and validates hexokinase type 4 (HK4), an isozyme of hexokinase in the liver and pancreas, as an important target of C2-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone (βdGT), a xanthone glucoside suggested to have antidiabetic property. In the study, we applied the computational pipeline of molecular docking followed by the molecular dynamics simulations to shortlist potential βdGT protein targets. The analysis of protein dynamics and the binding free energy (ΔG) led us to the identification of HK4 as a key βdGT target, whereby the binding mode and domain dynamics suggested the activator function of βdGT. βdGT bound to the allosteric site of the isozyme ～13?Å away from the substrate (glucose)-binding site. The binding free energy of the ligand-protein complex was energetically feasible (ΔG, –41.61?kcal/mol) and the cleft angle deviation between the two (small and large) domains of HK4 revealed differential HK4 dynamics in response to βdGT binding. 3D structure analysis of the isozyme-ligand complex highlighted the role of Arg63, Glu67 and Lys458 in ligand stabilization and hydrophobic interactions mediated by Tyr214 and Met235. Experimental validation of the results of computational analysis confirmed the activator function of βdGT on HK4. The study has implication in diabetes as βdGT may be used to lower the blood glucose level by activating hepatic and pancreatic hexokinase without the risk of hypoglycemia.

Communicated by Ramaswamy H. Sarma     

Sodium butyrate inhibits aerobic glycolysis of hepatocellular carcinoma cells via the c‐myc/hexokinase 2 pathway

Aerobic glycolysis is a well‐known hallmark of hepatocellular carcinoma (HCC). Hence, targeting the key enzymes of this pathway is considered a novel approach to HCC treatment. The effects of sodium butyrate (NaBu), a sodium salt of the short‐chain fatty acid butyrate, on aerobic glycolysis in HCC cells and the underlying mechanism are unknown. In the present study, data obtained from cell lines with mouse xenograft model revealed that NaBu inhibited aerobic glycolysis in the HCC cells in vivo and in vitro. NaBu induced apoptosis while inhibiting the proliferation of the HCC cells in vivo and in vitro. Furthermore, the compound inhibited the release of lactate and glucose consumption in the HCC cells in vitro and inhibited the production of lactate in vivo. The modulatory effects of NaBu on glycolysis, proliferation and apoptosis were related to its modulation of hexokinase 2 (HK2). NaBu downregulated HK2 expression via c‐myc signalling. The upregulation of glycolysis in the HCC cells induced by sorafenib was impeded by NaBu, thereby enhancing the anti‐HCC effect of sorafenib in vitro and in vivo. Thus, NaBu inhibits the expression of HK2 to downregulate aerobic glycolysis and the proliferation of HCC cells and induces their apoptosis via the c‐myc pathway.     

Effect of lentivirus-mediated shRNA inactivation of HK1, HK2, and HK3 genes in colorectal cancer and melanoma cells

Background

The switch from oxidative phosphorylation to glycolysis in proliferating cancer cells, even under aerobic conditions, has been shown first in 1926 by Otto Warburg. Today this phenomenon is known as the “Warburg effect” and recognized as a hallmark of cancer. The metabolic shift to glycolysis is associated with the alterations in signaling pathways involved in energy metabolism, including glucose uptake and fermentation, and regulation of mitochondrial functions. Hexokinases (HKs), which catalyze the first step of glycolysis, have been identified to play a role in tumorigenesis of human colorectal cancer (CRC) and melanoma. However, the mechanism of action of HKs in the promotion of tumor growth remains unclear.

Results

The purpose of the present study was to investigate the effect of silencing of hexokinase genes (HK1, HK2, and HK3) in colorectal cancer (HT-29, SW 480, HCT-15, RKO, and HCT 116) and melanoma (MDA-MB-435S and SK-MEL-28) cell lines using short hairpin RNA (shRNA) lentiviral vectors. shRNA lentiviral plasmid vectors pLSLP-HK1, pLSLP-HK2, and pLSLP-HK3 were constructed and then transfected separately or co-transfected into the cells. HK2 inactivation was associated with increased expression of HK1 in colorectal cancer cell lines pointing to the compensation effect. Simultaneous attenuation of HK1 and HK2 levels led to decreased cell viability. Co-transfection with shRNA vectors against HK1, HK2, and HK3 mRNAs resulted in a rapid cell death via apoptosis.

Conclusions

We have demonstrated that simultaneous inactivation of HK1 and HK2 was sufficient to decrease proliferation and viability of melanoma and colorectal cancer cells. Our results suggest that HK1 and HK2 could be the key therapeutic targets for reducing aerobic glycolysis in examined cancers.