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Molecular Endocrinology, doi:10.1210/me.2006-0356
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Molecular Endocrinology 21 (6): 1478-1487
Copyright © 2007 by The Endocrine Society

Molecular Coordination of Hepatic Glucose Metabolism by the 6-Phosphofructo-2-Kinase/Fructose-2,6- Bisphosphatase:Glucokinase Complex

W. Ed Smith, Sara Langer, Chaodong Wu, Simone Baltrusch and David A. Okar

Department of Veterans Affairs Medical Center (W.E.S., D.A.O.), Minneapolis, Minnesota 55417; Institute of Clinical Biochemistry (S.L., S.B.), Hannover Medical School, 30623 Hannover, Germany; and Department of Biochemistry (C.W.), Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455

Address all correspondence and requests for reprints to: David A. Okar, Ph.D., Veterans Affairs Medical Center, Minneapolis, 1 Veterans Drive, Room 3P-105, Minneapolis, Minnesota 55417. E-mail: David.Okar{at}med.va.gov.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucokinase (GK) and 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase (FBP-2) are each powerful regulators of hepatic carbohydrate metabolism that have been reported to influence each other’s expression, activities, and cellular location. Here we present the first physical evidence for saturable and reversible binding of GK to the FBP-2 domain of PFK-2/FBP-2 in a 1:1 stoichiometric complex. We confirmed complex formation and stoichiometry by independent methods including affinity resin pull-down assays and fluorescent resonance energy transfer. All suggest that the binding of GK to PFK-2/FBP-2 is weak. Enzymatic assays of the GK:PFK-2/FBP-2 complex suggest a concomitant increase of the kinase-to-bisphosphatase ratio of bifunctional enzyme and activation of GK upon binding. The kinase-to-bisphosphatase ratio is increased by activation of the PFK-2 activity whereas FBP-2 activity is unchanged. This means that the GK-bound PFK-2/FBP-2 produces more of the biofactor fructose-2,6-bisphosphate, a potent activator of 6-phosphofructo-1-kinase, the committing step to glycolysis. Therefore, we conclude that the binding of GK to PFK-2/FBP-2 promotes a coordinated up-regulation of glucose phosphorylation and glycolysis in the liver, i.e. hepatic glucose disposal. The GK:PFK-2/FBP-2 interaction may also serve as a metabolic signal transduction pathway for the glucose sensor, GK, in the liver. Demonstration of molecular coordination of hepatic carbohydrate metabolism has fundamental relevance to understanding the function of the liver in maintaining fuel homeostasis, particularly in managing excursions in glycemia produced by meal consumption.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE BIFUNCTIONAL ENZYME 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBP-2; Bif) is an important regulator of cellular fuel metabolism, especially in the liver (1, 2). Its primary function is to control the intracellular content of the biofactor fructose-2,6-bisphosphate (F-2,6-P2), a potent activator of 6-phosphofructo-1-kinase (PFK-1) (3). Activation of PFK-1 in vivo by increased hepatic F-2,6-P2 has been conclusively demonstrated (4, 5). In addition to confirming that increased hepatic F-2,6-P2 activates glycolysis and reduces glycemia, some reports also suggested that this metabolic regulator coordinates hepatic glycolysis with lipid metabolism (5). Predicated on yeast 2 hybrid and phage display results suggesting that liver PFK-2/FBP-2 bound to glucokinase (GK; hexokinase 4) (6) and using a graded overexpression of Ser-32-dephosphorylated rat liver PFK-2/FBP-2, Payne et al. (7) have demonstrated the retention of GK in the cytosol of mouse liver is promoted by the bifunctional enzyme. Serine-32-dephosphorylated hepatic PFK-2/FBP-2 is also a net producer of F-2,6-P2 and is generated from phospho-serine-32 bifunctional enzyme by the action of protein phosphatases that are stimulated by glucose via xyulose-5-phosphate (8) and/or insulin signaling (9). Conversely, PFK-2/FBP-2 is phosphorylated by protein kinase A, activated in response to glucagon via increased cellular cAMP (10).

GK, or hexokinase 4, also has a major role in regulating fuel metabolism. It has been described as a glucose sensor, primarily on the basis of its function in pancreatic ß-cells where it couples the abundance of postprandial serum glucose to the release of insulin (11). How GK functions as a glucose sensor in the liver has been less obvious because it has not been clear to which cellular response the GK signal is coupled. Regardless, because the glucose transporter 2 in hepatocytes is not strongly regulated, hepatic GK essentially controls glucose uptake, which suggests a more fundamental role than simply sensing glucose (12). Due to the role of the liver in maintaining systemic glucose homeostasis, perhaps it is not surprising that the orchestration of the complex metabolic responses to nutritional and hormonal signals requires the close association of two very powerful metabolic regulators such as GK and PFK-2/FBP-2. Yet, despite all of the in vivo evidence that these proteins work together, no direct evidence of their binding was detected (7, 13). This is an essential point as there are many ways in which proteins can interact without binding, especially on the time scale (days) required for studies, such as those of Payne et al. (7), which use adenovirus-mediated overexpression of cellular proteins. A more recent report by Garcia-Herrero et al. (14) has demonstrated pull down of 35S-labeled PFK-2/FBP-2 by affinity resin-bound GK from extracts of Hep2G cells, but even these crucial observations do not conclusively settle the issue of whether GK and PFK-2/FBP-2 directly interact.

For these reasons we set out to investigate the in vitro binding of GK to the FBP-2 domain and PFK-2/FBP-2. For this purpose we chose the Alexa Fluor (AF) series of dyes (Molecular Probes, Inc., Eugene, OR), which have very well-defined absorbance and fluorescence spectra and are available with a variety of reactive groups directed toward different moieties of the surface of the proteins. We used two amine-reactive AF dyes that also support fluorescent resonance energy transfer (FRET) with AF-488 as donor and AF-594 as acceptor. The nonradiative FRET is distinct from radiative transfer of fluorescent energy, which is due to the excitation of the acceptor by the light fluoresced by the donor, because it is a direct transfer of energy between the excited states (15). The FRET signal is detected as fluorescence intensity (FI) at the emission wavelength of the acceptor with excitation of the donor. FRET is inversely proportional to the sixth power of the distance between the donor and acceptor (15). The only way the dyes on different proteins can produce a FRET signal is if they bind to each other because these dyes must come within approximately 50 A to support FRET. Because we have not undertaken the protein engineering required to assure single-site labeling of these enzymes, the labeling reactions produce multiply labeled proteins, which severely impedes quantitative FRET studies aimed at determining the relative orientation of the proteins in the complex. Even so, by exploiting a qualitative use of FRET, we devised a series of experiments in which we titrated the FBP-2 domain (ND249) tagged with an acceptor fluorophore (AF-594) with GK tagged with a donor (AF-488). Because we measured the FI of the acceptor while exciting the donor, if FRET occurs during the titration, the FI will be limited by complex formation at high concentrations of titrant. In addition to reconfirming the weak binding, this method also determines the stoichiometry of the complex.

We also determined whether and how the binding of GK and PFK-2/FBP-2 affects their activities. These critical data highlight the switch-like properties of GK and PFK-2/FBP-2, which provide for at least a portion of the acute reversal of hepatic carbohydrate metabolism during the immediate response to refeeding after a fast. According to theory, each molecular contribution to biological switching adds with the others, until at the macroscopic level, a near step-function switch can be attained by a subset of cellular proteins (16, 17, 18). Whether such constitutes a molecular machine is a question for other publications, but we are not the first to suggest this possibility for the PFK-2/FBP-2 and F-2,6-P2 (19). By this analysis, the physical junction of these two biological switches, especially within the context of the in vivo studies, has a fundamental role in providing the kind of synergistic molecular interactions that, in sum, produce efficient biological switching within the liver.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GK Binds Specifically to the FBP-2 Domain of PFK-2/FBP-2
The fluorescent labeling of GK and ND249 (an N-terminal deletion of 249 amino acids from the rat liver bifunctional enzyme), the FBP-2 domain, allowed us to trace these proteins in nickel-nitrilotriacetic acid (Ni:NTA) resin pull-down assays using H6PFK-2/FBP-2 and H6GK, respectively. Figure 1AGo shows that including H6GK promotes the retention of ND249*594 (the * indicates fluorescent tag and the number identifies which AF dye was used) by the resin, indicating that GK binds to the FBP-2 domain. Furthermore, the ability of a 5-fold molar excess of unlabeled ND249 to elute the ND249*594 from the pellet (Fig. 1BGo) confirms that the binding is reversible. When the binding of ND249*488 to H6GK was challenged by proteins other than unlabeled ND249 (Fig. 1CGo), neither the glycolytic enzymes triose phosphate isomerase (TPI) and yeast hexokinase 1 (HK1), nor BSA could effectively wash ND249*488 from the complex, thus confirming that the binding of the FBP-2 domain to GK is specific. The binding of GK to the intact bifunctional enzyme was assessed by the pull-down assay summarized in Fig. 2Go, where H6PFK-2/FBP-2 was shown to bind to GK*594 or GK*488. With this set of experiments we have exhaustively assessed the GK:PFK-2/FBP-2 complex with each binding partner as the bait and the prey, while demonstrating that the interaction is specific.


Figure 1
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  		Fig. 1. Affinity Resin Pull-Down Assays

Panel A demonstrates that H6GK can pull down the liver FBP-2 domain, ND249*594, because more fluorescent protein was eluted from the resin when the bait was included (solid bar) relative to when it was not (open bar). Panel B shows that a 5-fold molar excess of unlabeled ND249 displaced the ND249*594 from the resin (gray bar). The data are background corrected, i.e. the hatched bar is the difference between the bars in panel A and accounts for the nonspecific binding of ND249 to the resin. All data are averages over three experiments conducted using the same stock proteins. Panel C reiterates that ND249 (gray bar) washes ND249*488 (hatched bar) from the pellet and the control proteins (open bars) do not. Competitor proteins were: TPI, yeast hexokinase 1 (HK1), and BSA. n = 9 for hatched and gray bars; n = 3 for the open bars. The SEM is indicated for each bar and the P values were calculated by Student’s t test. Result: ND249 binding to GK is reversible and saturable. NA, Not added; Arb., arbitrary.

 

Figure 2
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  		Fig. 2. Affinity Resin Pull-Down Results

Panels A and B show results using two differently labeled GK preparations. Open bars are nonspecific binding and solid bars are from samples including the bait. The average FI determined for each fluorophore over four determinations are reported with SEM; indicated P values were calculated by Student’s t test.. Result: GK binds to PFK-2/FBP-2. NA, Not added; Arb., arbitrary.

 
The Molar Stoichiometry of the Complex Is 1:1
Because the pull-down assays suggested that the binding of PFK-2/FBP-2 and GK is a relatively weak interaction, we sought to both reconfirm its formation and determine the stoichiometry by titrating ND249*594 with GK*488 while observing the emission spectrum of the acceptor (AF-594) produced by exciting the donor (AF-488), as shown in Fig. 3Go, A and B. Initially, we adjusted the conditions of the amine-reactive labeling reactions to minimize the dye-protein ratio (data not shown), but these did not provide sufficient sensitivity to reproducibly detect protein binding, so we completed the studies with fluorescently labeled proteins (GK*488 and ND249*594) with dye-to-protein ratios from 3–4. The representative fluorescence spectra in Fig. 3Go, A and B, show enhanced FI in the mixed samples. This excess fluorescence intensity (XSFI) may be seen more clearly by subtracting the spectra as detailed in Materials and Methods. Representative difference spectra for a complete titration are given in Fig. 3CGo. Note the similarity of the two highest GK*488 points, suggesting saturation. By plotting XSFI at 621 nm as a function of GK*488 concentration, as in Fig. 3DGo, the nonlinear result suggests that some portion of the energy transferred between the fluorophores is due to FRET. Because the donor and acceptor must be rather close in space to accomplish FRET, these data confirm the formation of a GK:FBP-2 complex. The representative data in Fig. 3DGo were acquired by titrating a 5 µM sample of ND249*594 with GK*488. The titration curve plateaus near the 1:1 molar stoichiometry, thus determining the stoichiometry of the complex.


Figure 3
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  		Fig. 3. FRET Titration of ND249*594 with GK-488

Panels A and B are representative raw data showing the fluorescence emission spectra from the microplate: 1) mixed sample; 2) GK*488 alone; and 3) ND249*594 alone. The labels in panel D indicate where the data in panels A and B fall in the titration curve. At each point, the amount of GK*488 (traces 1 and 2) is the same, whereas that of ND249*594 is constant over all points (trace 3). The difference spectra (see Equation 1Go) for an entire titration series is given in panel C. The FRET titration curve is plotted in panel D, which clearly shows a plateau near a 1:1 molar stoichiometry. The average over four determinations ± SEM are reported with a spline fit. Arb., Arbitrary.

 
Molecular Coordination of Hepatic Glucose Metabolism
We hypothesized that formation of the GK:PFK-2/FBP-2 complex, in and of itself, serves to increase both the activity of GK and the kinase-to-bisphosphatase ratio of PFK-2/FBP-2. To test this hypothesis we investigated whether the activities of the enzymes were influenced by formation of the protein-protein complex. When GK activity was monitored as a function of PFK/2/FBP-2, as shown in Fig. 4Go, a significant activation of GK was observed at a 10-fold molar excess of PFK-2/FBP-2. When purified GK was added to PFK-2/FBP-2 in vitro, no significant change in PFK-2 or FBP-2 activities was detected. Considering that the interaction we were investigating had previously been predicted based on the results of in vivo studies, we reasoned that a putative missing component exists in the cell that serves to facilitate the binding of the proteins, thus increasing the relative population of the bound state. Therefore, we obtained extracts of mouse livers from 16-h fasted animals and 30-min post bolus glucose infusion as mimic of the fed state, and we used these in hopes of adding back the missing component(s). The extracts were prepared to preserve total protein. Each extract was diluted to 1 mg/ml total protein with extract buffer. These diluted fed liver extracts contained more F-2,6-P2 than did those fasted livers (1.77 ± 0.03 pM in fed liver extracts vs. undetectable in fasted liver extracts), which reflects the hepatic response to refeeding. These values are lower than typically reported because the method we used to prepare the liver extracts was not optimal for preservation of F-2,6-P2, which requires extraction with alkali (20).


Figure 4
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  		Fig. 4. GK Is Activated by PFK-2/FBP-2

The specific activity of GK is increased by addition of PFK-2/FBP-2 at equimolar and 10-fold molar excess. The increase attains significance at the 1:10 point. The averages over four experiments are reported ± SEM.

 
The absolute PFK-2 activity of endogenous PFK-2/FBP-2 in mouse liver extracts was low, and the specific activity decreased as more extract was included in the assays, as shown in Fig. 5AGo. These demonstrated no significant correlation with nutritional state. This result is, perhaps, not surprising because the concentration of PFK-2/FBP-2 in the extracts is low, and previous studies have shown that it must be partially purified by polyethylene glycol (PEG) precipitation to clearly reveal the effects of changing nutritional state. We interpret the data in Fig. 5AGo as the dilution of some PFK-2 inhibitory component of the liver extracts that relieves inhibition more effectively when it is more highly diluted, i.e. when smaller aliquots of the extracts are used. The nature of this putative inhibitor remains unknown. Experimentally we could not use less than 10 µl of the extracts, because although the specific activity was high, the absolute activities were quite low. Interestingly, the addition of purified GK to liver extracts produces higher PFK-2 activity, as shown in Fig. 5Go, B and C, for fed and fasted extracts, respectively. Addition of the GK dialysis buffer did not activate the endogenous PFK-2, nor did the GK stock exhibit any PFK-2 activity (data not shown). The paired t test is justified because we included the extract controls in each assay. Of the eight total conditions, five are significantly different to at least 90% probability, three to greater than 95%. The increased PFK-2 activity in the presence of GK was more prominent for fasted liver extracts (Fig. 5CGo) relative to fed (Fig. 5BGo), but essentially the effect of GK on the PFK-2 activity was independent of the nutritional state. Paired Student’s t tests using all data for each nutritional state, regardless of the amount of extract assayed, gave P = 0.006 and 0.001 for fed and fasted extracts, respectively. Thus, the activation of endogenous PFK-2 in liver extracts by added GK is significant, yet correlates only slightly with nutritional state. The trend is toward more significant activation of extract PFK-2 by GK in the fasted state, perhaps reflecting the loss of GK upon fasting, but this suggestion has many caveats. When proteins other than GK were added to fed and fasted liver extracts (Fig. 6Go, A and B), no activation of endogenous PFK-2 was observed; in fact, addition of TPI, HK1, or BSA appeared to facilitate loss of PFK-2 activity in the extracts, again independent of nutritional state. The addition of protein kinase A inhibitor (PKI) to the liver extracts did not activate PFK-2 either, suggesting that changes in the posttranslational modification state of PFK-2/FBP-2 are not occurring during our assays. Little influence of GK on FBP-2 activity in either PFK-2/FBP-2 or ND249 was observed in vitro. Because the liver extracts quenched the fluorescence of oxidized nicotinamide adenine dinucleotide phosphate (NADP)+ that is the read-out of the FBP-2 assay, we were precluded from investigating the effect of these on FBP-2 activity.


Figure 5
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  		Fig. 5. PFK-2 in Liver Extracts Is Activated by Added GK

The specific PFK-2 activity in liver extracts from 16-h fasted (gray bars) and fed (infused with a glucose bolus of 2 g/kg body weight over 30 min) (open bars) mouse livers are shown in panel A. The P value reported in panel A is unpaired, whereas all others are from paired t tests. Panels B (fed extracts) and C (fasted extracts) show that when 5 µM GK was added (solid bars) to either liver extract, the PFK-2 was significantly activated in five of eight conditions. When buffer was substituted for the added GK, no activation was detected. Data reported are the averages over all assays at each amount of extract ± SEM. n = 12 for fasted extracts and 14 for fed.

 

Figure 6
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  		Fig. 6. PFK-2 in Liver Extracts Is Not Activated by Adding Proteins Other Than GK

The open bars in either panel are the extracts diluted with buffer. A, Fed liver extracts with either BSA (gray bars) or HK1 (solid bars) added to the extracts. B, Fasted liver extracts with either TPI (gray bars) or PKI (solid bars) added to the extracts. n = 6 for open bars; n = 3 for all others. No statistically significant differences were observed at any point.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Based on the results of these studies we conclude that GK binds reversibly to bifunctional enzyme at a site in the FBP-2 domain to form a 1:1 stoichiometric complex. Moreover, the pull-down assays demonstrate that when the proteins were mixed in equimolar proportions, the extent of complex formation is essentially the same whether we used PFK-2/FBP-2 or the ND249 bisphosphatase domain as binding partner for GK. This localizes the GK binding site to the FBP-2 domain, consistent with the prediction of Baltrusch et al. (6). This is the first evidence of direct binding between GK and PFK-2/FBP-2 proteins. Because the bifunctional enzyme functions as a dimer, this implies the heterotetrameric structure, GK2:Bif2, for the full complex. In contrast, because the ND249 is monomeric, a dimeric structure, GK:ND249, is implied. Within the context of the in vivo studies (7), our results confirm direct binding of GK and PFK-2/FBP-2 as a mechanism by which these two enzymes interact within the hepatocyte. Very recently, this interaction has been observed in the human hepatoma cell line Hep2G by affinity resin pull down of 35S-labeled PFK-2/FBP-2 by GK (14), yet those crucial studies did not preclude the participation of another protein in the complex. This is an important point because the interaction of PFK-2/FBP-2 with the scaffolding protein 14-3-3 has been reported (21).

Our most interesting observations are the enzyme kinetic data that suggest formation of the PFK-2/FBP-2:GK complex activates GK and PFK-2, whereas the FBP-2 is, apparently, unchanged. The data in Figs. 5Go must be interpreted with care, however, due to the inclusion of live liver extracts in the PFK-2 assays. The specific PFK-2 activities in various amounts of the liver extracts (Fig. 5AGo) demonstrate that the more dilute the extract, the higher the specific activity. These data formalize long-standing anecdotal observations that PFK-2 activity is inhibited by some component in cell extracts. This observation does not undermine any previous reports about the activities of PFK-2/FBP-2 from tissue or cell lysates because the bifunctional enzyme was routinely concentrated and partially purified by PEG precipitation in those studies. Here we sought to maintain all components of the extracts because we sought to complement the interaction between GK and PFK-2/FBP-2. The observed effect of dilution upon PFK-2 specific activity compelled us to assure that when we added purified GK to the extracts (closed bars in Fig. 5Go) the extent of dilution was exactly the same as in the blanks (open and gray bars in Fig. 5Go). This same condition also applied to the addition of control proteins to the extracts (Fig. 6Go). None of these control proteins showed any activation of PFK-2, reiterating that the effect of GK is specific and significant. Moreover, our inclusion of PKI in the control experiments suggests that changes in the phosphorylation state of PFK-2/FBP-2 are not at the root of the activation of PFK-2 by GK. It is surprising, perhaps, that we see no significant correlation of these effects with the nutritional state. This may be related to the nature of the live liver extracts, which are not optimal for assay of the bifunctional enzyme due to the lack of precipitation by PEG, which would be incompatible with our purposes in these studies. These caveats leave open the question of which particular component(s) in the liver extracts promote the activation of PFK-2 by GK. Taken together with the observed weak binding between GK and PFK-2/FBP-2, the PFK-2 activation data suggest at least two possibilities: 1) activation of PFK-2 by binding of GK requires some other component in the liver extracts; or 2) some component in the liver extracts promotes binding of GK to PFK-2/FBP-2. Although the former cannot be conclusively eliminated, the latter is consistent with weak binding in vitro, and published observations that suggest the in vivo binding is more extensive (6, 7, 13, 14). Conversely, the activation of GK by binding of PFK-2/FBP-2 (Fig. 4Go) does not require the presence of liver extracts. The requirement of a 10-fold molar excess of PFK-2/FBP-2 is consistent with weak binding, because such implies a low population of the bound state.

The binding of GK to PFK-2/FBP-2 both increases the phosphorylation of glucose and the net synthesis of F-2,6-P2, implying a molecular coordination of two sequential steps in hepatic glucose metabolism that directly couple serum glucose to glycolysis, as depicted in Fig. 7Go. We do not mean to suggest that the liver is primarily responsible for the clearance of excess blood glucose, because the role of skeletal muscle in this regard has been well established. At the same time, we cannot discount the possibility that the liver is responsible for some glucose disposal, as the early work of Wu et al. (22) suggested that the reduction in serum glucose was closely associated with increasing hepatic F-2,6-P2 and lactate content, even before the effect of adenoviral-mediated overexpression of PFK-2/FBP-2 was maximal. Moreover, the 1[13C]glucose magnetic resonance spectroscopy study of Choi et al. (23) clearly detected a more active PFK-1 in the livers of living mice that were overexpressing a mutant PFK-2/FBP-2 that produced chronically elevated hepatic F-2,6-P2. At the same time, if our observation of weak binding between GK and PFK-2/FBP-2 is reflected in vivo, it may argue against the GK/PFK-2/FBP-2 complex contributing quantitatively to the increase in metabolic flux from serum glucose to glycolysis because it implies a relatively low population of the bound state. However, it may be that another factor in the liver promotes binding of GK to PFK-2/FBP-2, increasing the population of the bound state. At the same time, the reported effects of elevated hepatic F-2,6-P2 on gene expression (24), GK translocation (7), and Akt phosphorylation state in mouse liver (25) suggest that the binding of bifunctional enzyme to GK may elicit profound regulatory effects even if it cannot quantitatively increase hepatic glucose disposal. From this perspective, the glucose-sensing capabilities of GK are adapted to the regulation of liver metabolism by interaction with PFK-2/FBP-2.


Figure 7
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  		Fig. 7. Schematic Model of the Role of GK:PFK-2/FBP-2 in Hepatic Metabolism

Formation of the complex directly couples increased glucose phosphorylation uptake in the liver with the commitment to glycolysis via F-2,6-P2. G-6-P, Glucose-6-phosphate; F-6-P, fructose-6-phosphate; F-1-P, fructose-1-phosphate; GLUT2, glucose transporter 2; GKRP, GK regulatory protein.

 
The molecular coordination revealed by our experiments is very interesting, because it serves to amplify the switch-like behavior implied by the translocation of GK in response to the regulatory dephosphorylation of Ser-32 in PFK-2/FBP-2 (3, 26). As others have pointed out, the ability of a biological system to exhibit switch-like properties, such as those that underlie the acute hepatic response to refeeding, is no small feat (18, 27). The theoretical discussion of Hervagault and Canu (28) suggests that bistability underpins fast biological switching, and it has been suggested that these can be built up by the synergistic interaction of many different mechanisms. It is clear that GK, PFK-2/FBP-2, and F-2,6-P2 work together to provide one component of the biological switch that allows the liver to accomplish the acute response to refeeding after a fast. The GK regulatory protein is clearly a major player in this switching mechanism, and it is only brevity that precludes its inclusion in our discussion (29). Therefore, our results underscore the potential of targeting hepatic F-2,6-P2 content as a desirable point of intervention to ameliorating the perturbations of carbohydrate and lipid metabolism that are associated with obesity and diabetes. We have also identified a new target for the design of novel pharmaceuticals because stabilizing the GK:PFK-2/FBP-2 complex will increase hepatic glucose disposal.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Experimental Animals
No animals were killed for these studies. All liver extracts obtained were from excess sample generated by Dr. Wu in the course of his work at the University of Minnesota. Accordingly, all protocols were reviewed and approved by the Institutional Animal Care and Use Committees at the University of Minnesota.

Expression and Purification of Proteins
All proteins were expressed from plasmid vectors in Escherichia coli (BL21DE3, M15, and XK1Blue) and subsequently purified by either the histidine-binding resin Ni:NTA affinity chromatography (for His-tagged;H6 proteins), strepavidin (for Strep-Tag PFK-2/FBP-2), or slight modification of established chromatographic methods (for non-H6 proteins) (30, 31, 32). We modified the purification methods for non-H6 proteins by implementing them on the BioCAD Perfusion Chromatography System and POROS resins, which increased the yield and minimized the time required for protein production. SDS-PAGE and standard kinetic assays were used to assess the quality of the purified proteins and all data reported were derived from fresh samples of at least 90% purity (see supplemental Figs. 1 and 2 published on The Endocrine Society’s Journals Online web site at http://mend. endojournals.org). We have produced six different proteins for use in these studies, i.e. human liver GK and H6GK, rat liver PFK-2/FBP-2, H6PFK-2/FBP-2, Strep-Tag PFK-2/FBP-2, and ND249. The ND249 bisphosphatase domain is an N-terminal deletion of 249 amino acids from the rat liver bifunctional enzyme sequence that has been extensively studied by x-ray crystallography and nuclear magnetic resonance spectroscopy. Proteins were expressed from either pET 3a, pET 16b, pASK-IBA7, or pQE30, plasmids for ND249, PFK-2/FBP-2, H6PFK-2/FBP-2, Strep-Tag PFK-2/FBP-2, and H6GK, respectively [Novagen (Madison, WI) or QIAGEN (Chatsworth, CA)]. The pLysS system was used for expression of PFK-2/FBP-2 (Novagen). Sequence comparisons for GK and PFK-2/FBP-2 between human and rat liver show extensive identity and almost complete homology; therefore, the difference in species should not influence the results or conclusions.

Labeling Proteins with Extrinsic Fluorophores
Only non-H6 proteins were labeled with AlexaFluor (AF) amine-reactive fluorophores. Purified proteins were exchanged into the labeling buffer and reacted with either Alexa Fluor-488, or AF-594 according to the protocols supplied by the manufacturer (Molecular Probes). After the labeling reactions were complete, the unreacted dye and other small molecule reaction products were removed by desalting and dialysis into the storage and binding buffers. Dyes were chosen to support FRET with AF-488 as donor and AF-594 as acceptor (R0 = 60). Because the fluorophores are well characterized and the extinction coefficients known, the amount of these in the protein samples can be determined and then compared with the amount of protein as determined by the Coomassie dye binding assay (Bio-Rad Laboratories, Inc., Hercules, CA).

Affinity Resin Pull-Down Assays
The nickel chelating resin, Ni:NTA, was used to pull down proteins with the six histidine (H6) affinity tag fused to the N terminus. All experiments were performed by adding samples containing the proteins to small vials containing wet Ni:NTA resin equilibrated in PBS (10 mM phosphate; 2.7 mM KCl; 137 mM NaCl, pH 7.4). To probe for binding of GK to the FBP-2 domain of PFK-2/FBP-2, the H6 proteins were used as bait to pull the fluorescently labeled binding partners from solution. The results were revealed by assaying for the fluorescent protein after eluting the bait H6-proteins with 500 mM imidazole in the M5 microplate reader by acquiring the pertinent emission spectrum of the AF dye used. H6GK was used to pull down ND249*594 (ND249 tagged with AF-594) and to probe for reversibility by including a 5 molar excess of unlabeled ND249. Control experiments were conducted in exactly the same way, but substituted either TPI, HK1, or BSA for the unlabeled ND249. In a separate experiment, H6PFK-2/FBP-2 was used to pull down either GK*594 or GK*488.

All pull-down experiments were conducted in PBS by the following protocol. First, 75 µl of a well-suspended, charged, 50% Ni:NTA resin slurry was dispensed into small vials. These were centrifuged very lightly, and the supernatant was quantitatively removed by pipet. The resin was equilibrated with fresh binding buffer (250 µl), which was removed by centrifugation and pipetting. The protein samples were premixed (total volume 250 µl) from concentrated protein stocks and binding buffer. Although the absolute amount of protein added varied from assay to assay, the molar ratio of GK to either PFK-2/FBP-2 or ND249 was typically 1, although in a few cases a 2–3 molar excess of GK was used. Protein concentrations ranged from 200 pM to 1.2 µM in the samples before they were added to the resin. Blanks were prepared using the dialysate from the H6 protein samples in place of the proteins themselves. After approximately 15 min incubation at room temperature, the mixed samples were added to the equilibrated resin, mixed gently, incubated for approximately 10 min, and centrifuged, and the supernatant was removed and placed in the wells of a 96-well microplate. The pellets were quickly washed with binding buffer (200 µl), and the wash was also placed in the microplate. The H6 proteins were eluted from the Ni:NTA resin with 500 mM imidazole in the binding buffer, and the eluants were placed in the same microplate. The fluorescence in the samples was measured with a Microdynamics (Sunnyvale, CA) M5 plate-reader using excitation at 488 or 594 nm and emission at 519 nm and 617 nm, for AF-488 and AF-594, respectively. Samples were prepared in triplicate for each determination, and each plate was read three times.

FRET Titration
The ability of fluorophores to transfer fluorescence energy nonradiatively is a function of the spectral parameters of the fluorophores, their relative orientation, and the distance between them. The choice of amine-reactive AF-488 and AF-594 in these studies determines the first two of these, because the overlap constant is determined by the donor acceptor pair (R0 = 60 in this case), and the attachment to amines, primarily lysine groups, implies that the fluorophores can move relative to the protein to which they are linked. The intensity of the transferred fluorescence at the emission wavelength for the acceptor is inversely proportional to the sixth power of the distance between the fluorophores (15). In the present report, because the multiple fluorophores are randomly attached to the proteins, detailed measurement of the putative FRET would not likely reveal much structural information.

Instead we used these labeled proteins to qualitatively determine the stoichiometry of the GK:PFK-2/FBP-2 complex. By acquiring the emission spectra at 1 nm resolution (excitation of AF-488 at 470 nm) from samples containing identical amounts of ND249*594 (acceptor) and increasing amounts of GK*488 (donor) while exciting at 470 nm (Microdynamics M5), we were, in effect, able to titrate ND249*594 with GK*488. We used 20 mM Tris, 100 mM NaCl, 2 mM dithiothreitol (pH 7.4) for the FRET titrations. On the same microplate we included samples of ND249*594 with the dialysate from GK*488 sample preparation added in place of GK*488 and GK*488 with dialysate from ND249*594 sample preparation in place of ND249*488. In practice we lowered the excitation wavelength to 470 nm to reduce direct excitation of AF-594. All samples were mixed in small vials, after which 200–250 µl was pipetted into the wells. The XSFI was calculated by subtracting the spectra according to Equation 1Go:

Formula 1(1)
The XSFI 621 nm was then plotted as function of the GK*488 added. If the proteins form a discrete complex, then the XSFI at 621 nm is due to nonradiative transfer (FRET) and will be limited by complex formation producing a nonlinear plot that plateaus at high concentrations of GK*488. Any influence of inner-filter effects are nominal because the absorbance of the excitation light by the acceptor is identical in all samples because the concentration of ND249*594 is the same in every sample and is taken into account by Equation 1Go. Absorbance of the fluoresced light by the acceptor is precluded by the relative lack of absorbance for AF-488 at 621 nm.

Significantly, we designed these experiments so that the titration range covers the substoichiometric through GK molar excess based on our hypothesis of a 1:1 complex. Furthermore, we prepared samples in an array with the dimensions determined by the number of GK concentrations and the three conditions, i.e. GK*488 alone, ND*594 alone, and the mixed protein samples. In this way, we had multiple determinations of the ND249*594 in each assay, and these worked as internal controls that provide a measure of the inherent scatter in the data introduced by sample handling and inhomogeneities in the wells. All XSFI values reported, although small relative to the absolute FI measured, are much larger than the range of FI at 621 nm observed between samples of the fluorescently labeled proteins alone and so are significant changes in the emission of the acceptor (data not shown). Because we were able to prepare concentrated protein stock solutions of GK*488 and ND249*594, we could match the protein concentrations between FRET titrations. Based on the fluorescence emission spectra of the ND249*594 and the low population of the bound state due to the weak binding suggested by the pull-down assays and a plethora of negative evidence from biophysical methods for assessing protein-protein binding that are predicated on tight binding and predominant bound states, we used relatively high protein concentrations titrating 5 µM ND249*594 with GK*488. We tried to prepare fluorescently labeled PFK-2/FBP-2 but found very low labeling, likely due to the relative paucity of expressed protein (relative to GK and ND249) and the difficulty in preparing highly concentrated protein samples of PFK-2/FBP-2 for use in the labeling reactions.

Liver Extracts
Male C57BL mice (12 wk old) were obtained from the Jackson Laboratory (Bar Harbor, ME). After an overnight (16-h) fast, the mice were given an ip bolus injection of saline (fasted) or glucose solution at a dose of 2 g/kg body weight (fed) over 30 min. Mice were immediately euthanized, and the livers were collected, as described previously (25). Fresh liver extracts were prepared in homogenizing buffer containing 20 mM TES (pH 7.8), 1 mM dithiothreitol, 100 mM KCl, 5 mM EDTA, 5 mM EGTA, 1.2 mM phenylmethylsulfonylfluoride, and 2.5 mg/liter leupeptin. Total protein concentrations were quantified using the BCA reaction (Pierce Chemical Co., Rockford, IL), or Coomassie Dye Binding (Bio-Rad). Extracts were aliquoted, flash frozen, and kept at –70 C until use in the PFK-2 assays.

Enzyme Kinetics
The kinetic assays of GK and PFK-2/FBP-2 are well established and have long been the interest of the authors. Here, we have adapted the fluorometric FBP-2 assay of Tominaga et al. (33) to the use of microplates and the M5 plate reader, whereas the assays of GK and PFK-2 were conducted according to published methods (34, 35). H6GK was incubated for 60 min without and with PFK-2/FBP-2 in a 1:2 and 1:10 ratio in GK assay buffer (20 mM HEPES; 125 mM KCl; 7.5 mM MgCl2*6H2O; 5 mM Na-ATP; 0.5 mM NADP; 0.5 U/ml glucose-6-phosphate-dehydrogenase). One unit of enzyme activity was defined as 1 mol glucose-6-phosphate formed from glucose and ATP per min at 37 C. Enzyme activity was expressed as units per mg cellular protein. The plate-based fluorometric FBP-2 assay was accomplished by adding a reaction mix (200 µl) containing (50 mM Tris, pH 7.5; 5 mM MgCl2; 50 µM NADP; 15 µM F-2,6-P2; 0.4 µg glucose-6-phosphate dehydrogenase; and PGI) to 10 µl of each sample predispensed into the wells of a microplate and subsequent reading of the FI due to reduced nicotinamide adenine dinucleotide (Ex = 354 nm; Em = 452 nm) produced as a monitor of substrate hydrolysis (33). The PFK-2 activity is determined by following the change in F-2,6-P2 over time, where the F-2,6-P2 is determined by the Van Shaftingen method using potato PPi:PFK-1 (36) prepared in house by the protocol of Wessberg et al. (37) adapted to the BioCAD and POROS resin. The PFK-2 reaction mix contains 20 mM Tris (pH 7.5), 5 mM ATP, 5 mM F-6-P, 10 mM MgCl2, and 5 mM Pi. All activities are expressed as picomoles of F-2,6-P2 produced per min per mg of total protein. Determination of the PFK-2 activity in the mouse liver extracts was complicated by the presence of F-2,6-P2 in the extracts, as well as that of any other compound that might influence PFK-2 activity over the 10–15 min needed to determined changes in F-2,6-P2. Addition of GK, or the control proteins (TPI, HK1, BSA, and PKI), to the liver extracts was made from concentrated stock solutions. The final concentration of the added proteins was the same, because we compensated for less extract by addition of extraction buffer.


   ACKNOWLEDGMENTS
 
We thank Dr. Gülin Öz at the University of Minnesota for constructive comments during the preparation of the manuscript.


   FOOTNOTES
 
This work was supported by Department of Veterans Affairs, Minnesota Medical Foundation, and the Veterans Affairs Post Fund.

Disclosure: W.E.S., S.L., C.W., and S.B. have nothing to declare. D.A.O. has previously consulted for Novo Nordisk.

First Published Online March 20, 2007

Abbreviations: AF, Alexa Fluor; FBP-2, fructose-2.6-bisphosphatase; FI, fluorescence intensity; F-2,6-P2, fructose-2,6-bisphosphate; FRET, fluorescent resonance energy transfer; GK, glucokinase; HK1, yeast hexokinase 1; NADP, nicotinamide adenine dinucleotide phosphate; Ni:NTA, nickel-nitrilotriacetic acid; PEG, polyethylene glycol; PFK-1, phosphofructo-1-kinase; PFK-2, 6-phosphofructo-2-kinase; PKI, protein kinase A inhibitor; TPI, triose phosphate isomerase; XSFI, excess fluorescence intensity.

Received for publication August 28, 2006. Accepted for publication March 13, 2007.


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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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