Therefore, it is not surprising that glucose catabolism glycolysis is elevated in many cancers, particularly those with the most rapid growth rates Warburg et al. It is also not surprising that the elevated consumption of glucose by such cancers is utilized clinically worldwide both as a diagnostic tool via 18 F-deoxy glucose positron emission tomography 18 FDG-PET and as a prognostic marker.
Critical to this highly glycolytic phenotype is the first enzymatic step of glucose phosphorylation Bustamante and Pedersen, ; Bustamante et al. Here, glucose is entrapped by phosphorylation for the tumor's utility, an event that involves specific isozymes of hexokinase HK and how they are expressed, regulated, and localized within the tumor in a manner that is highly advantageous to the tumor but destructive to the host Pedersen, ; Mathupala et al.
Hexokinases catalyse the essentially irreversible first step of the glycolytic pathway below where glucose is phosphorylated to glucosephosphate with concomitant de-phosphorylation of ATP. Examination of the primary sequence of each of the four isoforms implicate those with a high affinity for glucose, that is, HK I, II, and III, as arising via duplication of an ancestral glucokinase gene similar to that which encodes HK IV Tsai and Wilson, , , ; Ardehali et al.
There is also evidence that a similar set of events takes place, at least in part, when normal pancreatic islet cells become tumors Vischer et al. Interestingly, most normal mammalian tissues express very little HK II with those from muscle, adipocytes and lung expressing low but significant levels Wilson, , Although HK II is a major player in helping maintain the highly malignant state, it has four other key protein partners Figure 1.
The net result of cooperation among these four proteins is the rapid and efficient production of glucosephosphate that serves not only as the precursor for glycolysis but also for biosynthesis of key metabolites via the pentose-phosphate pathway and the mitochondrial tricarboxylic acid cycle, both essential for the growth and proliferation of cancer cells.
But this is not all! In an ingenious example of the cancer cell's efficiency in sustaining its own life long enough to proliferate and metastasize, it instructs the binding of HK II to VDAC and likely other proteins for another purpose. This is to inhibit mitochondrial-induced apoptosis and suppress cell death. Delivery of glucose and ATP to hexokinase HK II bound to the outer mitochondrial membrane within a malignant cell and metabolic fates of the glucosephosphate GP formed.
Glucose brought across the plasma membrane by glucose transporters 1 is rapidly phosphorylated by HK II 5 bound to voltage-dependent anion channel VDAC 4 located on the outer mitochondrial membrane. To maintain the highly glycolytic metabolic flux of such malignant cells, the product GP is rapidly distributed across key metabolic routes.
The primary routes are a direct entry of the GP into the pentose-phosphate shunt for biosynthesis of nucleic-acid precursors and b conversion of the GP via the glycolytic pathway to pyruvate and lactic acid. Here, whereas the lactic acid is transported out on lactate transporters 6 to provide an unfavorable environment for surrounding normal cells, some pyruvate is directed to mitochondria via the pyruvate transporter 7 , to provide substrates for the tri-carboxylic acid TCA cycle.
Citrate produced by this cycle then exits the mitochondria on the citrate transporter 8 to help synthesize membrane components phospholipids and cholesterol that are essential for tumor proliferation. Discussed below are the key enzymes and transporters that help support the pivotal role of HK II in promoting the malignant phenotype.
In order to satisfy the enhanced glucose metabolism of malignant tumors, their plasma-membrane glucose uptake requirements need to be met. Glucose uptake in mammalian tissues is achieved by a set of five transmembrane transporters Pauwels et al. Similar to the HK isoforms, the Glut isoforms also differ in their transport kinetics. Increased glucose transport in malignant tumors has been associated with increased and deregulated expression of these transporters, mostly with over-expression of the Glut-1 isoform.
In human tumors, a high level of Glut-1 expression has been associated with poor prognosis Macheda et al. As Glut expression at the cell-surface is mediated by hormone-induced cycling of transporter vesicles between intracellular pools and the cell membrane, disregulated trafficking may contribute also to an enhanced display of Glut on malignant tumors thus facilitating enhanced glucose uptake Smith, Finally, much more needs to be learned about the role of Gluts in highly malignant tumors as it relates to how the key transporter s involved deliver glucose to HK II bound to VDAC of the mitochondrial outer membrane.
Figure 1 suggests that the glucose must diffuse through quite a distance, an unlikely scenario. Considering that highly malignant cancer cells and the mitochondria within them are not static, but likely dynamic, it does not seem unreasonable to suggest that the Glut on the cell membrane and HK II bound to VDAC on mitochondria may come into contact. The first detailed indications of a close relationship between mitochondria and HK were revealed over two decades ago Rose and Warms, , ; Bustamante and Pedersen, ; Bustamante et al.
Significantly, the latter study by Nakashima et al. Voltage-dependent anion channel is now known to exist in several different isoforms that are abundantly expressed and localized in the outer membrane of eukaryotic mitochondria Blachly-Dyson et al. Voltage-dependent anion channels form the primary channel for movement of adenine nucleotides through the outer membrane, and serve as the mitochondrial binding site for both HK and glycerol kinase, among other mitochondria-bound proteins.
Enhanced expression of VDAC on tumor mitochondria in comparison to normal tissue has also been observed Shinohara et al. Although several isoforms of VDAC are known, they do have similar kinetic characteristics, which indicate that the contribution of VDAC to enhanced HK binding and glucose phosphorylation is due to quantitative differences in binding site availability Shinohara et al. Most accessory proteins that modulate apoptosis via their interactions with mitochondria also use VDAC-1 as their anchor.
In the latter case, the opened VDAC is proposed to restore metabolic exchange across the outer mitochondrial membrane while preventing the release of cytochrome c Vander Heiden et al.
With regard to the enhanced glycolysis in tumors, an alternate view has also been put forward, where suppression of mitochondrial function owing to closure of porins is proposed to be responsible Lemasters and Holmuhamedov, According to this hypothesis, HK binding to VDAC inhibits its conductance and thus suppresses mitochondrial function while stimulating glycolysis.
However, this view makes it difficult to explain the well-observed phenomenon of direct access of mitochondrial generated ATP to the VDAC-bound HK Arora and Pedersen, , and thus needs further examination. The respective HK isozymes and transporter isoforms that are implicated in enhancing glucose phosphorylation with concomitant upregulation of glycolysis in malignant tumors are encoded at different chromosomal loci.
Tumors harness a multitude of genetic, epigenetic, transcriptional and post-translational strategies for enhanced expression and function of hexokinase HK II. During tumorigenesis of tissues where HK II is absent, the gene may be first brought out of its hibernation by demethylation, and then amplified 5—fold. Subsequently, the highly promiscuous promoter of the gene, which is activated by HIF-1, p53, glucose, and by both insulin and glucagon, further facilitates the tumor's requirements regardless of the nutritional status of the tumor-bearing host, and fuels the enhanced and continued synthesis of the gene product.
In contrast to other hexokinase isoforms, HK II harbors two active sites per enzyme moiety. As much as a fold amplification of the enzyme may be observed in malignant tumors owing to these different processes. Recently, epigenetic events leading to activation or silencing of alternate HK gene isoforms have been described during hepato-carcinogenesis. Methylation restriction endonuclease analysis of normal hepatocytes and hepatoma cells has indicated differential methylation patterns in the HK II gene promoter during tumorigenesis where the HK isozyme expression shifts from HK IV, that exhibits a low affinity for glucose, to the HK II and HK I that exhibit a high affinity for glucose Goel et al.
Thus, these observations indicate that one of the initial events in activating the HK II gene during either transformation or tumor progression may reside at the epigenetic level. A first indication that gene amplification plays a role in enhanced expression of HK isozymes with a low K m for glucose high apparent affinity during tumorigenesis was demonstrated with Southern blot analysis and fluorescence in situ hybridization of hepatocytes and a hepatoma cell line.
Here, enhanced HK II expression was observed to be associated with at least a fivefold amplification of the HK II gene relative to that of normal hepatocytes Rempel et al. This gene amplification was located intra-chromosomally, and most likely occurs at the site of the resident gene. No rearrangement of the gene was detected. Thus, these findings revealed that gene amplification plays a key role in overexpression of the low K m high apparent affinity HK II isoform in a highly malignant tumor expressing the high glycolytic phenotype.
Whether this will prove to be the case in other highly malignant tumors, or whether such tumors have devised multiple strategies for assuring enhanced glucose utilization, remains to be established. It also controls the levels of glycogen synthesis in hepatocytes of the liver. Hexokinase Interactions Through specific binding to a porin or voltage dependent anion channel, hexokinases I and II can associate physically to the outer surface of the external membrane of the mitochondria.
This gives hexokinase direct access to ATP that the mitochondria generates. Mitochondrial hexokinase can be observed in elevated amounts, up to times more than in normal tissues, in growing malignant tumor cells.
Hexokinase deficiency causes Chronic Haemolytic Anaemia, which is caused by a mutation to the HK gene. This gene codes for the HK enzyme and the mutation leads to less HK activity, which causes hexokinase deficiency.
Shipped with Ice Packs Add to Cart. Back to Top. Glucokinase has one active binding site for glucose and one for ATP, which is the energy source for phosphorylation. This active binding site is located between the small and large domains. The carboxyl terminus is part of the alpha 13 helix, which codes for the region that forms half of the binding site for glucose.
Glucokinase can be modulated to form an inactive and active complex. The inactive conformation forms when the alpha 13 helix has been modulated away from the rest of the molecule forming a large space. This space is too large to bind glucose so it is said to be in the inactive form.
The alternative is when the alpha 13 helix is modulated to form a smaller space thus activating the protein [4]. Glucokinase includes the where glucose forms hydrogen bonds at the bottom of the deep crevice between the large domain and the small domain. E, E shown in green of the large domain, T, K shown in red of the small domain, and N, D shown in yellow of a connecting region form hydrogen bonds with glucose. The shows a different conformation. The again shows structural differences. The differences in these two conformations allows glucokinase to function properly in different levels of glucose concentration.
Proposed Mechanism for Glucokinase: As described above, glucokinase has a distinct conformation change from the active and inactive form. Experiments have also shown an intermediate open form based on analysis of the movement between the active and inactive form.
The switch in conformations between the active form and the intermediate is a kinetically faster step than the change between the intermediate and the inactive form. The inactive form of gluckokinase is the thermodynamically favored unless there is glucose present. Glucokinase does not change conformation until the glucose molecule binds. The conformation change may be triggered by the interaction between Asp and the glucose molecule.
Once glucokinase is in the active form, the enzymatic reaction is carried out with the presence of ATP. The experiments suggest that glucokinase is found in hepatocyte nuclei and are found inactive at low plasma glucose levels, but found active when higher glucose levels are present.
GKRP would then would likely be an allosteric inhibitor of glucokinase that specifically binds to the inactive form of glucokinase. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK's channeling GP towards glycolysis catabolic use , and cytoplasmic HKII regulating glycogen formation anabolic use.
Metabolic measurements suggest that HKI exclusively promotes glycolysis, whereas HKII has a more complex role, promoting glycolysis when bound to mitochondria and glycogen synthesis when located in the cytosol.
Glycogen breakdown upon glucose removal leads to HKII inhibition and dissociation from mitochondria, probably mediated by increases in glycogen-derived GP. These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, GP and Akt through regulation and subcellular translocation of HKII.
In contrast, HKI, which activity and regulation is much less sensitive to these factors, is mainly committed to glycolysis. This may be an important mechanism by which HK's allow cells to adapt to changing metabolic conditions to maintain energy balance and avoid injury.
This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. Upon entering the cell, glucose is phosphorylated to glucosephosphate GP , which is used catabolically in glycolysis, or anabolically in glycogen synthesis and lipid synthesis via the pentose phosphate shunt. In both cases, this first step is catalyzed by hexokinases HKs , which comprise a family of four isoforms. The expression of GLUT4 and HKII coincides with the development of insulin sensitivity as muscle switches from a straight carbohydrate to a mixed fat-carbohydrate diet [4].
In adult muscle, fatty acids as well as glucose and glycogen are available as substrates to support oxidative metabolism [5]. GP facilitates glycogen synthesis by reciprocally activating glycogen synthase GS and inhibiting glycogen phosphorylase GP [6] , [7] , and possibly by stimulating translocation of HK from mitochondria to the cytoplasm. However, it was not clear at that time how independent was the anabolic function of the Type II isozyme and binding to mitochondria were related.
Since then, Wilson and others [10] , [11] , [12] , [13] , [14] , [15] , [16] , [17] , [18] have shown that the interaction of HKs with mitochondria is not static, but is regulated by factors such as glucose, GP and kinases such as Akt and GSK Thus, a picture is emerging that HKII may play a dual role: channeling GP into the glycogen and the pentose phosphate pathways when localized in the cytoplasm, and preferentially shuttling GP to glycolysis and oxidative phosphorylation when bound to mitochondria [19].
In contrast, HKI generally facilitates glycolysis; although under some specific non-physiological conditions may contribute to glycogen synthesis [20]. In contrast, HKII serves primarily anabolic functions.
Our findings support the hypothesis that in response to changes in glucose, subcellular translocation of HKII dynamically directs the metabolic fate of glucose between catabolic glycolysis and anabolic glycogen synthesis and pentose phosphate shunt uses, while HKI remains associated with mitochondria to promote glycolysis. Factors such as GP and Akt play a central role in the regulation of HKII activity and localization in response to changes in glucose.
To test the hypothesis that HKI interacts preferentially with mitochondria to facilitate entry of GP into the glycolytic pathway, while HKII translocates into the cytosol to channel GP into the glycogen formation pathway, we carried out two types of optical imaging experiments in CHO cells and HEK cells. In Fig. S1 supports the interpretation that HKI, even when overexpressed, is predominantly bound to mitochondria.
CHO cells express low levels of glucose transporters GLUT , however, they have low rates of glucose uptake and metabolism compared to muscle cells [26]. We had previously shown that in native CHO cells, exposure to Cyto B prior to the addition of glucose fully blocked subsequent glucose uptake [26]. In contrast, Cyto B had almost no effect on the rate of glucose clearance following glucose removal Fig. S3A [1] vs. S3A [2].
S3B [4] , with a high rate of glucose transport uptake half-time 3. Thus, to study the metabolic fate of glucose in CHO cells overexpressing GLUTs, subsequent experiments were performed in the presence of Cyto B to block glucose efflux following glucose removal.
Data in Fig. These data clearly demonstrate that increased HKI activity, which is primarily associated with mitochondria, increases glucose metabolism, while increased HKII activity, which is found at least in part in the cytosol, slows glucose metabolism. Comparison of A and B indicates that HKI increases the rate of glucose clearance and thus metabolism, while comparison of A and C demonstrates that HKII has the opposite effect and decreases this rate. Panel E and F show images similar to those in Fig.
The bar graph in D quantifies these data. Thus, when glucose is removed at the end of the pulse, the cell mobilizes the newly synthesized glycogen for glycolysis, rather than metabolizing the residual intracellular glucose. This explanation is plausible only if glycogen mobilization also inhibits HK enzymatic activity, such that the conversion of residual intracellular glucose to GP is suppressed.
Since glycogenolysis involves production of GP, which inhibits HKs [21] , [22] , we postulated that elevated GP levels during glycogenolysis might be responsible for inhibiting HK activity, so that intracellular glucose clearance is delayed.
Consistent with this hypothesis, Fig. However, after a 75 s exposure to 10 mM glucose Fig. In contrast, exposure to 1 mM extracellular glucose for several minutes, which increased intracellular glucose level by less than half compared to 10 mM glucose, did not cause a delay in glucose metabolism Fig. Panel A illustrates how exposure to 10 mM extracellular glucose for 75 s 1 , 30 s 2 and 50 s 3 affects intracellular glucose clearance. In the 3 cases CytoB was applied for 15 s prior to removal of extracellular glucose.
The 3 rates are compared in the right hand side panel and show that the final rate of glucose clearance is similar in the 3 cases and that long exposure to extracellular glucose delays the clearance. In B a comparison of data obtained with 10 mM and 1 mM extracellular glucose show that application for up to two minutes of 1 mM glucose had no effect on the rate of intracellular glucose clearance, demonstrating that intracellular glucose must reach a threshold to induce this effect.
The right hand side panel shows again that the final rate of glucose clearance measured in the presence of 10 mM glucose is similar to the maximum rate measure with 1 mM and the effect of high glucose is thus to delay clearance. To test this hypothesis further, we imaged glycogen stores directly by using probes linking either mCherry or GFP to PTG and G L , both of which are part of the family of glycogen targeting subunits of PP-1 [27].
One day after transfection, cells were incubated for 2 to 3 hours in the absence of glucose to deplete glycogen stores. In the absence of GLUT1 overexpression, some CHO cells exhibited dim homogenous fluorescence, while others had a few small and bright punctuate deposits Fig. After re-addition of glucose 10 mM for 30—60 min, the number, size and brightness of small glycogen deposits began to increase. This phenomenon intensified over 24 hrs, until the deposits fused and partially filled the cell Fig.
After a two hour exposure, removal of glucose resulted in a rapid disappearance of glycogen deposits Fig. With overexpression of GLUT1, the rate of appearance of glycogen dramatically increased, such that small deposits were observed within a few minutes of exposure to 10 mM glucose Fig. In Panel A and D the cells were incubated in the absence of glucose for 2 to 3 hours prior to beginning cell imaging.
This incubation in the absence of glucose was carried out to deplete preformed glycogen. Without GLUT1 overexpression glycogen build up was slow, occurring over several hours.
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