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. 2011 Mar 9;6(3):e17674.
doi: 10.1371/journal.pone.0017674.

Subcellular localization of hexokinases I and II directs the metabolic fate of glucose

Scott John  1 , James N Weiss, Bernard Ribalet
Affiliations

Subcellular localization of hexokinases I and II directs the metabolic fate of glucose

Scott John et al. PLoS One. .

Abstract

Background: The first step in glucose metabolism is conversion of glucose to glucose 6-phosphate (G-6-P) by hexokinases (HKs), a family with 4 isoforms. The two most common isoforms, HKI and HKII, have overlapping tissue expression, but different subcellular distributions, with HKI associated mainly with mitochondria and HKII associated with both mitochondrial and cytoplasmic compartments. Here we tested the hypothesis that these different subcellular distributions are associated with different metabolic roles, with mitochondrially-bound HK's channeling G-6-P towards glycolysis (catabolic use), and cytoplasmic HKII regulating glycogen formation (anabolic use).

Methodology/principal findings: To study subcellular translocation of HKs in living cells, we expressed HKI and HKII linked to YFP in CHO cells. We concomitantly recorded the effects on glucose handling using the FRET based intracellular glucose biosensor, FLIPglu-600 mM, and glycogen formation using a glycogen-associated protein, PTG, tagged with GFP. Our results demonstrate that HKI remains strongly bound to mitochondria, whereas HKII translocates between mitochondria and the cytosol in response to glucose, G-6-P and Akt, but not ATP. 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 G-6-P.

Conclusions/significance: These findings show that the catabolic versus anabolic fate of glucose is dynamically regulated by extracellular glucose via signaling molecules such as intracellular glucose, G-6-P 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Subcellular distribution of HKI and HKII linked to YFP in CHO cells.
In the left panel HKI-YFP is expressed alone (A) or with HKII (B) (ratio 1∶3). In this case HKI-YFP is bound to mitochondria and is not displaced by overexpressed HKII. In the right panel HKII-YFP is expressed alone and shows a mixed distribution between mitochondria and cytosol (C). In this case overexpression of HKI with HKII-YFP (ratio 1∶3) prevents HKII-YFP interaction with mitochondria (D). Similar subcellular distribution of HKI-YFP and HKII-YFP was observed in HEK293 cells.
Figure 2
Figure 2. Effects of HKI and HKII on glucose utilization in CHO cells.
These data illustrates how overexpression of HKI (Panel B) and HKII (Panel C) affects glucose clearance measured in the presence of CytoB. 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. 1, which illustrate once more the interaction of HKI-YFP with mitochondria and the differential distribution of HKII-YFP, these differential distribution can be correlated with the glucose clearance data in B and C, respectively. The bar graph in D quantifies these data.
Figure 3
Figure 3. The time of exposure to extracellular glucose controls the rate of intracellular glucose clearance.
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.
Figure 4
Figure 4. Effects of GLUT1 overexpression on glycogen synthesis and breakdown in CHO cells.
The rate of glycogen formation without (A) and with (D) overexpression of GLUT1 and the rate of glycogen degradation (B) was studied using a protein targeted to glycogen linked to YFP (PTG-YFP). 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. This build up proceeded for up to 24 h filling almost completely the cell in some cases (C). With GLUT1 overexpression the rate of glycogen synthesis increased and glycogen deposits could be observed 5 min after exposure to 10 mM glucose outside. It is interesting to note that glycogen deposition occurred at least initially near the nucleus where mitochondria aggregate, suggesting that G-6-P generated by glycogen degradation may be directly fed on to mitochondria.
Figure 5
Figure 5. Overexpression of PTG delays glucose clearance.
Panels A and B obtained with CHO cells overexpressing GLUT1 illustrate how exogenous PTG delays glucose clearance. In both cases the cells were exposed to extracellular glucose for 50 s. Comparison of data in Panel B (overexpressed GLUT1) and C (no GLUT1 overexpression) illustrate the lack of effect of exogenous PTG in the absence of GLUT1 overexpression, supporting the hypothesis that GLUT1 is necessary for rapid build up of the glycogen store and inhibition of glucose utilization. Compilation of traces in D illustrates once more that the final rate of glucose clearance is similar in all cases and that increased glycogen synthesis only delays glucose utilization.
Figure 6
Figure 6. Removal of glucose causes translocation of HKII, but not HKI, into the cytosol.
Images in Panel A obtained with HKI-YFP expressed in CHO cells show that removal of glucose did not affect HKI interaction with mitochondria. In contrast, HKII begun to dissociate from mitochondria 5 min after glucose removal (B). Panel C shows the rates measured as ratio of fluorescence intensity obtained from intracellular domains without and with mitochondria. In almost all cases the region without mitochondria was selected at the cell periphery and that with mitochondria was selected near the nucleus. C1 is a comparison of the rates of HKI and HKII dissociation in response to glucose removal. C2 shows the rate of HKII reassociation with mitochondria after glucose readdition.
Figure 7
Figure 7. Constitutively active Akt prevents the effect of glucose removal on HKII dissociation from mitochondria.
Images in (A) obtained with overexpression of a constitutively active Akt illustrate how Akt prevents HKII dissociation evoked by removal of glucose. Panel B shows a quantification of this effect, comparing the rate of HKII dissociation from mitochondria in response to glucose removal in the presence and absence of exogenous Akt. Data in Panel C show how glucose clearance is affected by preincubation in the absence of glucose and how constitutively active Akt prevents this effect. Previous data (Fig. 6B) have shown that HKII dissociates from mitochondria 15 to 30 min after removal of extracellular glucose; comparison of traces C1 and C2 (in C2 cells were incubated for 30 min in the absence of glucose) indicates that this effect is accompanied by a decrease in glucose clearance. Thus, glucose-induced HK dissociation slows the rate of glucose utilization. The recording in C3 obtained with cells overexpressing constitutively active Akt indicates that Akt prevents the decrease in glucose clearance induced by incubation in the absence of glucose for 30 min. This effect is very likely related to the effect of Akt on HKII translocation. The bar graph in C4 quantifies the effects of glucose removal and Akt on glucose clearance.
Figure 8
Figure 8. Rates of association and dissociation of HKI and HKII in response to glucose, IAA and FCCP.
IAA, but not FCCP, facilitates HKII dissociation from mitochondria. Images in Panel A obtained with HKII-YFP expressed in CHO cells show that addition of the glycolysis inhibitor, IAA, in the presence of 10 mM glucose causes HKII dissociation from mitochondria within 5 to 15 min. In contrast, addition of FCCP, which depletes mitochondrial ATP, had no effect on HKII interaction with mitochondria (B). These data indicate that G-6-P, rather than ATP mediates the effect of glucose removal on HKII translocation. Panel C shows the rates measured as ratio of fluorescence intensity obtained from intracellular domains without and with mitochondria. C1 is a comparison of the rates of HKII dissociation from mitochondria in response to IAA and FCCP addition in the presence of 10 mM glucose outside. C2 illustrates the rate of reassociation HKII with mitochondria upon removal of IAA.
Figure 9
Figure 9. HKs dissociation from mitochondria in permeabilized cells.
Panels A and B illustrate the spontaneous dissociation of HKI-YFP in the absence and presence of G-6-P, respectively, after cell membrane permeabilization with 50 µM β-escin. Panel C1 and C2 show that HKI and HKII dissociate for mitochondria at very similar rates, and G-6-P increases these rates by approximately 4 fold (20 min vs. 5 min). The triangles represent measurements obtained with G-6-P (100 nM) and the circles are for values obtained in the absence of G-6-P. Panel C3 shows the change in intracellular G-6-P levels following glucose removal.
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