Interaction of 6-Phosphofructo-2-Kinase/Fructose-2,6- Bisphosphatase (PFK-2/Fbpase-2) with Glucokinase Activates Glucose Phospho

Home , Glucokinase

Interaction of 6-Phosphofructo-2-Kinase/Fructose-2,6- Bisphosphatase (PFK-2/FBPase-2) With Glucokinase Activates Glucose Phosphorylation and Glucose Metabolism in Insulin-Producing Cells Laura Massa,1 Simone Baltrusch,1 David A. Okar,2,3 Alex J. Lange,2 Sigurd Lenzen,1 and Markus Tiedge1

The bifunctional enzyme 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner he enzyme glucokinase (GK) plays a pivotal role for glucokinase (GK). Therefore, we studied the impor- in the recognition of glucose in pancreatic tance of this interaction for the activity status of GK ␤-cells and the regulation of glucose metabolism and glucose metabolism in insulin-producing cells by Tin the liver (1–7). In pancreatic ␤-cells, GK acts overexpression of the rat liver and pancreatic islet as a glucose sensor and catalyzes the rate-limiting step for isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overex- initiation of glucose-induced insulin secretion (6). GK is pression in RINm5F-GK cells significantly increased the regulated in a complex manner in pancreatic ␤-cells by GK activity by 78% in cells expressing the islet isoform, posttranslational modifications of the enzyme protein that by 130% in cells expressing the liver isoform, and by mainly depend on the intracellular glucose concentration 116% in cells expressing a cAMP-insensitive liver S32A/ (8–13). These posttranslational mechanisms of GK activity H258A double mutant isoform. Only in cells overex- regulation are comprised of conformational changes pressing the wild-type liver PFK-2/FBPase-2 isoform (14,15), sulfhydryl-group conversions (16–18), and inter- was the increase of GK activity abolished by forskolin, ␤ apparently due to the regulatory site for phosphoryla- actions with -cell matrix proteins (13,19), insulin gran- tion by a cAMP-dependent protein kinase. In cells over- ules (20,21), newly identified binding partners (22,23), and expressing any isoform of the PFK-2/FBPase-2, the GK-activating compounds (24). The hepatic GK regulatory increase of the GK enzyme activity was antagonized by protein, which binds and inhibits GK competitively and treatment with anti–FBPase-2 antibody. Increasing the confers short-term regulation of GK in the liver (25,26), is glucose concentration from 2 to 10 mmol/l had a signif- not expressed in pancreatic ␤-cells (13). Using a peptide icant stimulatory effect on the GK activity in phage display strategy, we recently identified the bifunctional RINm5F-GK cells overexpressing any isoform of PFK-2/ enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphos- FBPase-2. The interaction of GK with PFK-2/FBPase-2 phatase (PFK-2/FBPase-2) as a binding partner of the GK takes place at glucose concentrations that are physio- (23). PFK-2/FBPase-2 modulates intracellular levels of logically relevant for the activation of GK and the fructose-2,6-bisphosphate (F-2,6-P2), a highly potent regu- regulation of glucose-induced insulin secretion. This lator of carbohydrate metabolism (27–29). In liver, the new mechanism of posttranslational GK regulation may PFK-2/FBPase-2 enzyme is modulated by phosphorylation also represent a new site for pharmacotherapeutic in- tervention in type 2 diabetes treatment. Diabetes 53: and dephosphorylation, which significantly affects the 1020–1029, 2004 kinase/bisphosphatase ratio of this bifunctional enzyme (28,29). Rat pancreatic islets express the brain isoform of PFK-2/FBPase-2 (23), which does not appear to be regulated by cAMP-dependent protein kinases (PKAs) or phos- phatases at the NH2-terminus of the protein, as is the liver isoform (29). However, yeast two-hybrid studies clearly indicate that the GK protein interacts with the liver as well From the 1Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany; the 2Department of Biochemistry, Molecular Biology and as the islet PFK-2/FBPase-2 isoform (23). This raised the Biophysics, University of Minnesota, Minneapolis, Minnesota; and the 3VA question whether the PFK-2/FBPase-2–GK interaction par- Medical Center, Minneapolis, Minnesota. ticipates in the posttranslational regulation of GK enzyme Address correspondence and reprint requests to Dr. Markus Tiedge, Insti- tute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, activity. Knowledge of the physiological significance of GK Germany. E-mail: [email protected]. regulation through interaction with PFK-2/FBPase-2 may Received for publication 17 October 2003 and accepted in revised form 5 January 2004. have a central impact on the understanding of the failure L.M and S.B. contributed equally to this work. of ␤-cell function in type 2 diabetes. To elucidate the effect F-2,6-P2, fructose-2,6-bisphosphate; GK, glucokinase; MOI, multiplicity of of PFK-2/FBPase-2 binding to GK on GK enzyme activity infection; PKA, cAMP-dependent protein kinase; PFK-2/FBPase-2,6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. and glucose metabolism, the liver and islet PFK-2/FBPase-2 © 2004 by the American Diabetes Association. isoforms were overexpressed in the present study in

1020 DIABETES, VOL. 53, APRIL 2004 L. MASSA AND ASSOCIATES

RINm5F-GK and INS1 insulin-producing cells. The results er’s manual. The recombinant protein was characterized through SDS-PAGE show that overexpression of PFK-2/FBPase-2 resulted in a and activity measurements (42). The protein concentration was analyzed by a Bio-Rad protein assay. Then, 1 mg of FBPase-2 protein was diluted in 500 ␮l significant increase of GK activity in insulin-producing PBS and mixed with an equal volume of complete Freund’s adjuvant. Leghorn cells accompanied by corresponding increases in glucose hens were immunized by injecting the antigen into the pectoral muscle. Two metabolism. booster injections of 0.5 mg antigen mixed with incomplete Freund’s adjuvant were given 8 and 21 days later. Eggs collected 28 days after the first immunization were used for isolation of IgY from the yolk according to a RESEARCH DESIGN AND METHODS polyethylene glycol procedure (43). Serial dilutions of the IgY-FBPase-2 Materials. Restriction enzymes and modifying enzymes for the cloning polyclonal antibody were analyzed to detect the recombinant FBPase-2 procedures were from New England Biolabs (Beverly, MA) or Fermentas (St. protein and rat liver PFK-2/FBPase-2 by Western blot analysis. Leo-Rot, Germany). The SP6/T7 Transcription Kit and DIG Nucleic Acid Western blot analyses. Cells were homogenized by sonication in PBS (pH Detection Kit were obtained from Roche (Mannheim, Germany). Hybond 7.4), and insoluble material was pelleted by centrifugation. The protein nylon membranes were from Amersham (Braunschweig, Germany), and concentration was quantified by a Bio-Rad protein assay. Thereafter, dithio- Immobilon-P polyvinylidine difluoride membranes were from Millipore (Bed- threitol and bromophenol blue were added from concentrated stocks to yield ford, MA). The enhanced chemiluminescence detection system and autora- a final concentration of 100 mmol/l and 0.1%, respectively. Cellular protein (20 diography films were from Amersham. Forskolin was from ICN Biomedicals ␮g) was fractionated by reducing 10% SDS-PAGE and electroblotted to (Irvine, CA). All reagents of analytical grade were from Merck (Darmstadt, polyvinylidine difluoride membranes. The membranes were stained by Pon- Germany). All tissue culture equipment was from Gibco Life Technologies ceau to verify the transfer of comparable amounts of cellular protein. (Gaithersburg, MD). Nonspecific binding sites of the membranes were blocked by nonfat dry milk Tissue culture. RINm5F cells (30,31) overexpressing GK (RINm5F-GK cells) overnight at 4°C. GK immunodetection was performed as described previ- were generated by stable transfection of the human ␤-cell GK cDNA (32) in ously (13,18). For PFK-2/FBPase-2, the blots were incubated with the de- the pcDNA3 vector as described previously (13,33). Cells were grown in RPMI scribed FBPase-2 antibody at a dilution of 1:10,000, followed by a 2-h 1640 medium supplemented with 10 mmol/l glucose, 10% (vol/vol) FCS, incubation period with an anti-IgY peroxidase-labeled secondary antibody at a penicillin, streptomycin, and 250 ␮g/ml G418 in a humidified atmosphere at dilution of 1:40,000 at room temperature. The specific protein bands were

37°C and 5% CO2. INS1 cells (passage 80–90) were grown in RPMI 1640 visualized by chemiluminescence using the enhanced chemiluminescence medium supplemented with 10 mmol/l glucose, 10% (vol/vol) FCS, 2 mmol/l detection system and quantified by densitometry using the Gel-Pro Analyser L-glutamine, 1 mmol/l sodium pyruvate, 10 mmol/l HEPES, 50 ␮mol/l 2-mer- software. Linearity of the band intensities of the autoradiograms was verified captoethanol, penicillin, and streptomycin in a humidified atmosphere at 37°C by serial dilutions of recombinant ␤-cell GK or rat liver FBPase-2 protein, and 5% CO2 (34). respectively (data not shown). Stable overexpression of PFK-2/FBPase-2 in RINm5F-GK cells. PFK-2/ Assay of GK enzyme activity. GK activity measurements and GK Western FBPase-2 coding cDNAs for rat liver (35), rat liver S32A/H258A double mutant blot analysis were performed from identical samples to achieve a direct (36,37), and rat islet/brain (23,38) were subcloned as a HindIII-ApaI fragment comparison between GK protein expression and activity. The cells were into the pcDNA3-Zeo expression vector by standard molecular biology tech- homogenized in PBS (pH 7.4), and insoluble material was pelleted by niques (39). RINm5F-GK cells were transfected with the vector DNA by the centrifugation. GK enzyme activity was measured in soluble fractions by an use of CLONfectin (Clontech, Palo Alto, CA) as described in the manufactur- enzyme-coupled photometric assay consisting of glucose-6-phosphate dehy- er’s manual. Positive clones were selected through resistance against Zeocin drogenase, ATP, and NADPH (44). GK activity was determined by subtracting (250 ␮g/ml) and characterized further for PFK-2/FBPase-2 expression by the hexokinase activity measured at 1 mmol/l glucose from the activity Northern blot and Western blot analyses. In the present study, we used the measured at various glucose concentrations (1.6, 3.1, 6.3, 12.5, 25, and 100 RINm5F-GK–PFK-2/FBPase-2 islet clones I4 and I10, the liver clones L5 and mmol/l). Enzyme activity was expressed as units per milligram cellular L11, and the liver mutant clones LM11 and LM12. protein. One unit of enzyme activity was defined as 1 ␮mol glucose-6- Recombinant adenovirus preparation and transduction of RINm5F-GK phosphate formed from glucose and ATP per minute at 37°C. Vmax was and INS1 cells. The adenoviral vector system was provided by B. Vogelstein estimated on Hanes plots; S0.5 and Hill coefficients of GK were calculated from (Baltimore, MD) (40). Rat liver PFK-2/FBPase-2 coding cDNA was subcloned Hill plots (45). as a KpnI-EcoRV fragment in the pShuttle-CMV vector, and generation of the Determination of F-2,6-P2. RINm5F-GK cells were permeabilized by 10 recombinant adenoviral plasmid was performed by homologous recombina- ␮g/ml ␣-toxin from Staphylococcus aureus (Sigma, Taufkirchen, Germany) as tion with the pAdEasy-1 plasmid in Escherichia coli BJ5183 cells. Recombi- described in detail by Cascante et al. (46). F-2,6-P2 was determined in the nant adenoviruses were produced in 293 cells and purified by CsCl gradient supernatant by an enzyme-coupled method using pyrophosphate-dependent centrifugation as described before (40). The resulting virus containing the fructose-6-phosphate kinase (Sigma) and expressed as picomoles per milli- liver PFK-2/FBPase-2 is denoted AdPFK2L, and the virus containing only the gram DNA (47). CMV promoter without cDNA insert is denoted AdWT. RINm5F-GK cells and Glucose metabolism. The glucose oxidation rate was assessed as the 14 14 INS1 cells were transduced at a multiplicity of infection (MOI) of 5, 10, and 20 production of CO2 from D-[U- C]glucose. Glucose metabolism was mea- or 20, 40, and 80, respectively, for 2 h with stocks of either the AdWT sured as described previously (48) in batches of 1 ϫ 105 cells over a 1-h adenovirus or the AdPFK2L adenovirus. Transduced cells were incubated for incubation at 37°Cin40␮l Krebs-Ringer buffer containing different glucose 48 h in medium with 5% (vol/vol) FCS to reduce mitotic activity. The concentrations (0.5, 1, 2, 5, and 10 mmol/l). Total radioactivity added to the experiments were performed 48 h after viral transduction. Adenoviral infec- cells was 10 ␮Ci/ml, resulting in specific radioactivities of 18 Ϫ 1 Ci/mol tion did not affect the cell proliferation rate and did not induce cytopathic glucose. Cellular metabolism was stopped by the addition of 50 ␮l of 0.2 mol/l 14 ␮ effects in RINm5F-GK cells as well as in INS1 cells (data not shown). HCl, and the produced CO2 was captured by the addition of 100 l Northern blot analyses. Cells were lysed in a precooled buffered 4 mol/l 1-phenylethylamine. After1hat37°C, wells containing cells were removed, guanidine thiocyanate solution. Total RNA was isolated by a combined scintillation liquid was added, and the radioactivity was counted in a liquid 14 water-saturated phenol-chloroform-isoamyl alcohol extraction method (41). A scintillation spectrometer. Recoveries of externally added NaH CO2 were total of 5 or 10 ␮g RNA per lane was subjected to electrophoresis on checked routinely and used to correct the metabolic rates accordingly. denaturing formamide/formaldehyde 1% (wt/vol) agarose gels and transferred Statistical analysis. The data are expressed as means Ϯ SE. Statistical to nylon membranes. Hybridization with 11-DIG-UTP–labeled antisense cRNA analyses were performed by ANOVA followed by the Bonferroni’s test for probes and detection were performed as described previously (13,23). The multiple comparison (GK activity) or the Student’s t test (glucose oxidation) hybrids were visualized by chemiluminescence detection on a light-sensitive using the Prism analysis program (Graphpad, San Diego, CA). film for quantification by densitometry using the Gel-Pro Analyser software (Media Cybernetics, Silver Spring, MD). The data were normalized to the density of the 28S ribosomal bands (data not shown). RESULTS Generation of FBPase-2 antibody. The bisphosphatase domain of rat liver PFK-2/FBPase-2 (amino acid residues 250–470) was subcloned as a BamHI- Adenoviral overexpression of wild-type liver PFK-2/ SalI fragment into the pGEX-6P-1 vector (Amersham Pharmacia Biotech, FBPase-2 in RINm5F-GK and INS1 cells and its effect Freiburg, Germany). The glutathione-S-transferase (GST) Gene Fusion System on GK enzyme activity. Adenoviral infection resulted (Amersham Pharmacia Biotech) was used for the expression and purification of the FBPase-2 protein in Escherichia coli BL21 bacteria. The cleavage of the in a significant overexpression of the liver PFK-2/FBPase-2 glutathione-S-transferase tag was achieved by incubating the glutathione- enzyme in RINm5F-GK insulin-producing tissue culture sepharose column with PreScission protease as described in the manufactur- cells (Fig. 1A) as well as in INS1 insulin-producing tissue

DIABETES, VOL. 53, APRIL 2004 1021 GK REGULATION BY PFK-2/FBPase-2

FIG. 1. Adenoviral overexpression of rat liver PFK-2/FBPase-2 and its effect on GK enzyme activity in RINm5F-GK (A) and INS1 (B) cells. Cells were transduced for 2 h with wild-type AdWT (lanes 1, 3, and 5 and Ⅺ) or liver PFK-2/FBPase-2 AdPFK2L (lanes 2, 4, and 6 and f) adenoviral constructs using MOI 5, 10, and 20 (A) and MOI 20, 40, and 80 (B). The experiments were performed 48 h after the viral incubation. For Northern blot analyses, 10 ␮g of total RNA was loaded per lane. The blots were probed with antisense cRNA coding for rat liver PFK-2/FBPase-2 by nonradioactive hybridization. Shown are representative blots of four independent experiments. For Western blot analyses, 20 ␮g cellular protein was analyzed per lane by immunoblotting using a specific antibody against FBPase-2 or GK. Representative blots of four independent experiments are shown. GK activities were measured spectrophotometrically in cell extracts after sonication. Data are expressed as the percentage of enzyme activity measured in AdWT cells. Means ؎ SE from six to eight individual experiments are shown. *P < 0.05, **P < 0.01, ***P < 0.001 compared with enzyme activity of control AdWT cells (ANOVA/ Bonferroni’s test). culture cells (Fig. 1B). Using three different MOIs, gradu- INS1 cells, GK protein levels were 100/108% at an MOI of ally increasing levels of PFK-2/FBPase-2 expression were 20, 100/89% at an MOI of 40, and 100/105% at an MOI of 80 detectable in both Northern and Western blot analyses (average values from four experiments, AdWT-infected (Fig. 1). RINm5F-GK and INS1 cells showed endogenous control cells/AdPFK2L-infected cells) (Fig. 1). GK gene expression of PFK-2/FBPase-2 mRNA and protein, which expression was also not affected (data not shown). Thus, was detectable after longer exposure times of the blots the overexpression increased the GK activity on the post- (data not shown). The increase of the PFK-2/FBPase-2 translational level. mRNA and protein expression was accompanied by a Effects of PFK-2/FBPase-2 overexpression on glucose significant increase of GK enzyme activity by 40–150% in metabolism in RINm5F-GK and INS1 cells. RINm5F-GK both cell lines (Fig. 1). In control experiments, adenoviral and INS1 cells were infected with AdPFK2L at MOIs, infection of the cells with the AdWT virus did not affect the which resulted in an optimal activation of GK activity abundance of PFK-2/FBPase-2 mRNA and protein. Densi- (Figs. 1 and 2). At 48 h after viral infection, glucose tometric analysis confirmed that the level of GK protein metabolism was assessed by the glucose oxidation rate expression remained unchanged in the PFK-2/FBPase-2 (Fig. 2). As expected, from the glucose sensor concept of 14 14 overexpressing cells at all MOIs, irrespective of the signif- GK, the production of CO2 from D-[U- C]glucose in- icant increase of the GK enzyme activity (Fig. 1). In creased in a concentration-dependent manner in both RINm5F-GK cells, GK protein levels were 100/113% at an RINm5F-GK and INS1 cells. Cells with an activated GK MOI of 5, 100/89% at an MOI of 10, and 100/108% at an MOI after overexpression of the liver PFK-2/FBPase-2 showed of 20 (average values from four experiments, AdWT- significantly higher glucose oxidation rates of 50% infected control cells/AdPFK2L-infected cells) (Fig. 1). In (RINm5F-GK) and 100% (INS1) at all glucose concentra-

FIG. 2. Effect of rat liver PFK-2/FBPase-2 overexpression on the glucose oxidation rate in RINm5F-GK and INS1 cells. Cells were treated for 2 h with wild-type AdWT (E) or liver PFK-2/FBPase-2 AdPFK2L (F) adenoviral constructs using MOI 10 (RINm5F-GK) and MOI 40 (INS1). After 48 h, cells were incubated for 60 min at 37°C in Krebs-Ringer buffer without glucose. Thereafter, glucose metabolism was measured in the presence of various glucose concentrations (0.5, 1, 2, 5, and 10 mmol/l). Glucose oxidation was calculated from ؎ 14 the production of CO2. Means SE from four to six individual experiments are shown. *P < 0.05, **P < 0.01 compared with the glucose oxidation rate in control AdWT cells (Student’s t test).

1022 DIABETES, VOL. 53, APRIL 2004 L. MASSA AND ASSOCIATES

bisphosphatase activity ratio (36,37). All clones showed a significant increase of PFK-2/FBPase-2 expression on both the mRNA and protein level (Fig. 3). In parallel with the level of PFK-2/FBPase-2 overexpression, there was a significant increase in the GK enzyme activity to 148 and 178% in clones I10 and I4, to 155 and 230% in clones L5 and L11, and to 130 and 216% in clones LM11 and LM12, respectively (Fig. 3). Northern blot analyses and densitometric quantification revealed that overexpression of PFK-2/ FBPase-2 did not affect the GK mRNA levels in RINm5F-GK cells (Fig. 3). Comparable to the adenoviral overexpression of PFK-2/FBPase-2 (Fig. 1), the level of GK protein expression remained unchanged in all six RINm5F clones overexpressing PFK-2/FBPase-2 (Fig. 3). In RINm5F-GK cells stably overexpressing PFK-2/FBPase-2, GK protein levels were 100% in control cells, 106% in the I10 clone, 95% in the I4 clone, 101% in the L5 clone, 91% in the L11 clone, 99% in the LM11 clone, and 92% in the LM12 clone (average values from four experiments) (Fig. 3). Therefore, the activation of GK enzyme activity was apparently the result of a posttranslational regulation. The data indicate that the activation of GK by PFK-2/FBPase-2 also occurs with the islet-specific isoform. The most intriguing explanation for the activation of the GK was an allosteric activation by F-2,6-P2. However, the cellular Ϯ levels of F-2,6-P2 were 20 4 pmol/mg DNA in FIG. 3. Stable overexpression of PFK-2/FBPase-2 and its effect on GK Ϯ enzyme activity in RINm5F-GK cells. Analyses were performed in RINm5F-GK control cells, 20 3 pmol/mg DNA in the I4 control RINm5F-GK cells (C), RINm5F-GK–PFK-2/FBPase-2 islet islet PFK-2/FBPase-2 clone, 6 Ϯ 1 pmol/mg DNA in the L11 clones I10 and I4 (lanes 1 and 2), RINm5F-GK–PFK-2/FBPase-2 liver Ϯ clones L5 and L11 (lanes 3 and 4), and RINm5F-GK–PFK-2/FBPase-2 liver PFK-2/FBPase-2 clone, and 31 4 pmol/mg DNA in liver mutant clones LM11 and LM12 (lanes 5 and 6). For PFK-2/ the LM12 liver mutant PFK-2/FBPase-2 clone (means Ϯ SE FBPase-2 Northern blot analyses, 10 ␮g total RNA was loaded per lane. from seven independent measurements at 10 mmol/l glu- The blots were probed with antisense cRNA coding for rat liver or rat islet PFK-2/FBPase-2 by nonradioactive hybridization. Representative cose). Thus, the increase of GK activity brought about by blots of four independent experiments are shown. For GK Northern overexpression of PFK-2/FBPase-2 was not due to an blot analyses, 5 ␮g total RNA was loaded per lane. The blots were probed with antisense cRNA coding for rat islet GK by nonradioactive allosteric activation of the enzyme by F-2,6-P2. hybridization. Representative blots of three independent experiments PFK-2/FBPase-2 overexpression promotes glucose are shown. For Western blot analyses, 20 ␮g cellular protein was oxidation in RINm5F-GK cells. Rates of glucose oxida- analyzed per lane by immunoblotting using a specific antibody against 14 14 FBPase-2 or GK. Representative blots of four independent experi- tion, measured as production of CO2 from D-[U- C]glu- ments are shown. For measurement of GK activity, cells were homog- cose, increased in a concentration-dependent manner in enized by sonication and enzyme activities were measured spectrophotometrically. Data are expressed as the percentage of en- RINm5F-GK cells overexpressing the islet as well as the zyme activity measured in control RINmF5-GK cells (100%). Means ؎ liver and the liver mutant PFK-2/FBPase-2 isoforms. In SE from six to eight individual experiments are shown. *P < 0.05, each case, they were significantly higher than in ***P < 0.001 compared with enzyme activity in control cells (ANOVA/ Bonferroni’s test). RINm5F-GK control cells not overexpressing PFK-2/ FBPase-2 (Fig. 4A–C). Theoretically, the stimulatory effect tions in comparison to control cells treated with the on glucose metabolism in PFK-2/FBPase-2–overexpress- wild-type adenovirus (AdWT) (Fig. 2). Thus, the activation ing RINm5F-GK cells could be explained by allosteric of GK by PFK-2/FBPase-2 effectively coupled millimolar activation of the PFK-1 by F-2,6-P2. However, in control glucose concentrations to glycolysis as a prerequisite for experiments, it was demonstrated that RINm5F cells, in metabolic stimulus-secretion coupling. To delineate the which high-affinity hexokinase activities dominate the mechanism by which PFK-2/FBPase-2 activated the GK glucose phosphorylation, did not show any changes in the enzyme, we generated stable RINm5F-GK clones overex- glucose oxidation rate in the micromolar and millimolar pressing PFK-2/FBPase-2 isoforms with different kinetic concentration range after stable overexpression of liver and regulatory characteristics. PFK-2/FBPase-2 (data not shown). Thus, the increase of Stable overexpression of PFK-2/FBPase-2 in glucose metabolism in PFK-2/FBPase-2 overexpressing RINm5F-GK cells. After transfection of different iso- cells was conferred by the activity status of the GK enzyme forms of PFK-2/FBPase-2 into RINm5F-GK cells, we se- and may be independent of F-2,6-P2. lected overexpressing clones for the rat islet/brain isoform Effect of forskolin on GK enzyme activity in PFK-2/ (Fig. 3; clones I4 and I10), the liver isoform (Fig. 3; clones FBPase-2 overexpressing RINm5F-GK cells. The sig- L5 and L11), and a liver mutant lacking the phosphoryla- nificant increase of GK enzyme activity (of 155 and 226%) tion sites for PKA (Fig. 3; clones LM11 and LM12). The in cell clones L5 and L11 stably overexpressing the liver islet/brain isoform of PFK-2/FBPase-2 reflects the situation PFK-2/FBPase-2 isoform was nearly completely abolished in insulin-producing cells, whereas the liver mutant of through addition of forskolin (10 ␮mol/l) to the incubation PFK-2/FBPase-2 is insensitive to regulation of the kinase/ medium (94 and 132%) (Fig. 5A). Forskolin affected the

DIABETES, VOL. 53, APRIL 2004 1023 GK REGULATION BY PFK-2/FBPase-2

FIG. 4. Effect of PFK-2/FBPase-2 overexpression on the glucose oxidation rate in RINm5F-GK cells. Measurements were performed in RINm5F-GK–PFK-2/FBPase-2 islet clone I4 (A), RINm5F-GK–PFK-2/ FBPase-2 liver clone L11 (B), and RINm5F-GK–PFK-2/FBPase-2 liver mutant clone LM12 (C)(F), compared with RINm5F-GK control cells (E). Cells were grown overnight at 10 mmol/l glucose. After 48 h, cells FIG. 5. Effect of forskolin on GK enzyme activity in RINm5F-GK cells were incubated for1hat37°C in Krebs-Ringer buffer without glucose. overexpressing PFK-2/FBPase-2. Measurements were performed in Thereafter, glucose metabolism was measured in the presence of RINm5F-GK–PFK-2/FBPase-2 liver clones L5 and L11 (A), RINm5F- various glucose concentrations (0.5, 1, 2, 5, and 10 mmol/l). Glucose GK–PFK-2/FBPase-2 islet clones I10 and I4 (B), and RINm5F-GK–PFK- ؎ 14 oxidation was calculated from the production of CO2. Means SE 2/FBPase-2 liver mutant clone LM11 and LM12 (C). Cells were grown from five individual experiments are shown. *P < 0.05, **P < 0.01, overnight at 10 mmol/l glucose. Thereafter, cells were incubated for 2 h ***P < 0.001 compared with the glucose oxidation rate in control cells in the presence of 10 ␮mol/l forskolin. Finally, cells were homogenized (Student’s t test). by sonication and enzyme activities were measured spectrophotometrically. Data are expressed as the percentage of enzyme activity measured in untreated RINm5F-GK control cells. Means ؎ SE from six to activity ratio of the liver PFK-2/FBPase-2. Because F-2,6-P2 ten individual experiments are shown. *P < 0.05, ***P < 0.001 com- levels in cell clones L5 and L11 significantly decreased pared with enzyme activity in untreated control cells; ###P < 0.001 compared with enzyme activity in the absence of forskolin (ANOVA/ after forskolin treatment, it is likely that the activities of Bonferroni’s test). the bifunctional enzyme were shifted toward the FBPase-2 because of phosphorylation by a PKA. This inhibitory effect of forskolin (10 ␮mol/l) was also confirmed in significant increase of GK enzyme activity in the cell clone experiments on RINm5F-GK cells transfected with the LM12 overexpressing the liver mutant PFK-2/FBPase-2 liver PFK-2/FBPase-2 isoform adenoviral vectors (data not isoform, which lacks a regulatory phosphorylation site and shown). thus a key component of the bisphosphatase active site of In contrast, forskolin (10 ␮mol/l) did not abolish the this bifunctional enzyme (Fig. 5B and C) (29,36,37). significant increase of GK enzyme activity in the cell Effect of glucose on GK enzyme activity in PFK-2/ clones I10 and I4 overexpressing the islet PFK-2/FBPase-2 FBPase-2 overexpressing RINm5F-GK cells. An in- isoform. Forskolin (10 ␮mol/l) did also not abolish the crease of the glucose concentration in the incubation

1024 DIABETES, VOL. 53, APRIL 2004 L. MASSA AND ASSOCIATES

FIG. 7. Kinetic characteristics of GK in RINm5F-GK cells overexpressing PFK-2/FBPase-2. Measurements were performed in cellular extracts from RINm5F-GK–PFK-2/FBPase-2 islet clone I4 (F), RINm5F- GK–PFK-2/FBPase-2 liver clone L11 (f), and RINm5F-GK control cells FIG. 6. Effect of glucose on GK enzyme activity in RINm5F-GK–PFK-2/ (E). Cells were grown overnight at 10 mmol/l glucose. Thereafter, cells FBPase-2 cells. Measurements were performed in RINm5F-GK–PFK-2/ were homogenized by sonication, and GK enzyme activity was deter- FBPase-2 islet clone I4, RINm5F-GK–PFK-2/FBPase-2 liver clone L11, mined spectrophotometrically in the presence of the indicated concen- and RINm5F-GK–PFK-2/FBPase-2 liver mutant clone LM12, compared trations of glucose. Data are expressed in units per milligram cellular .with RINm5F-GK control cells. Cells were grown overnight at 10 mmol/l protein. Means ؎ SE from four individual experiments are shown glucose. Thereafter, cells were incubated for4hat2or10mmol/l glucose. Finally, cells were homogenized by sonication, and enzyme activities were measured spectrophotometrically. For Western blot FBPase-2 antibody (Fig. 8). In RINm5F-GK control cells, analyses, 20 ␮g cellular protein was analyzed per lane by immunoblot- which do not overexpress PFK-2/FBPase-2, an inhibitory ting using a specific antibody against GK. Representative blots of three effect of the anti–FBPase-2 antibody on GK enzyme activ- independent experiments are shown. Data are expressed as the percentage of enzyme activity measured in RINm5F-GK control cells in the ity was not observed (Fig. 8). Thus, the interaction of GK presence of 10 mmol/l glucose. Means ؎ SE from four to six individual with the bisphosphatase domain of PFK-2/FBPase-2 was a experiments are shown. *P < 0.05, ***P < 0.001 compared with enzyme prerequisite for the activation of the enzyme. activity in the presence of 2 mmol/l glucose (ANOVA/Bonferroni’s test). DISCUSSION medium from 2 to 10 mmol/l increased the GK enzyme The GK enzyme is regulated in a complex manner on both activity significantly in control RINm5F-GK insulin-pro- the transcriptional and posttranslational level. Posttrans- ducing cells (Fig. 6). Overexpression of the islet (clone I4) lational mechanisms of GK regulation play a pivotal role in as well as the liver (clone L11) and the liver mutant (clone the adaptation of the enzyme activity to the physiological LM12) PFK-2/FBPase-2 forms resulted in a threefold needs of the nutritional status in both liver and pancreatic greater stimulatory effect of glucose on GK enzyme activ- ␤-cells. In pancreatic ␤-cells, glucose is a key regulator of ity when the glucose concentration was increased from 2 GK enzyme activity by a currently unknown mechanism. to 10 mmol/l (Fig. 6). The stimulatory effect of PFK-2/ There is evidence that the GK interacts with intracellular FBPase-2 overexpression on GK enzyme activity was due structures in insulin-producing cells providing an indica- to a significant increase of the Vmax values to 222% (islet tion for an enzyme activity regulation through interaction isoform) and 236% (liver isoform), whereas the S0.5 values with specific proteins (13,21,22). In a systematic random were not significantly different to those of RINm5F-GK peptide phage display library screening, we recently iden- control cells (Fig. 7, Table 1). The S0.5 values for glucose tified the bifunctional enzyme PFK-2/FBPase-2 as a GK were somewhat lower than those in pancreatic islets and binding partner (23). PFK-2/FBPase-2 was transiently and liver presumably because of the high activity levels of low- stably overexpressed in RINm5F-GK and INS1 insulin- Km hexokinases in RINm5F-GK cells, which apparently producing cells to address the functional relevance of this interfere with the kinetic measurements of GK. Overex- interaction as it relates to the regulation of the GK enzyme pression of liver PFK-2/FBPase-2 resulted in a significantly activity. higher cooperativity for glucose with a Hill coefficient of Overexpression of PFK-2/FBPase-2 significantly in- 2.6 (Table 1). Notably, the level of GK protein expression creased GK enzyme activities in both RINm5F-GK and was not affected by the overexpression of PFK-2/FBPase-2 INS1 cells. The overexpression of PFK-2/FBPase-2 stimu- (Fig. 6). The GK protein levels were 100% at 2 mmol/l and lated GK enzyme activity at the posttranslational level, 107% at 10 mmol/l glucose in control cells, 101% at 2 whereas GK protein levels remained unchanged in com- mmol/l and 95% at 10 mmol/l glucose in the I4 clone, 101% parison to control cells. The GK activation in PFK-2/ at 2 mmol/l and 96% at 10 mmol/l glucose in the L11 clone, FBPase-2 overexpressing cells resulted in a higher glucose and 100% at 2 mmol/l and 101% at 10 mmol/l glucose in the oxidation rate, demonstrating that increased GK activity is LM12 clone (average values from three experiments). coupled with an increased metabolism of glucose in Effect of the anti–PFK-2/FBPase-2 antibody on GK insulin-producing cells (Fig. 2). It should be mentioned in enzyme activity in PFK-2/FBPase-2 overexpressing this context that in insulin-producing cell lines, glucose RINm5F-GK cells. The significant increase of GK enzyme metabolism is partly determined by high-affinity hexoki- activity in RINm5F-GK cells after overexpression of the nase isoenzymes, which are expressed at considerable islet (clone I4) as well as the liver (clone L11) and the liver levels. Thus, in these model cells the kinetics of the mutant (clone LM12) PFK-2/FBPase-2 forms was also glucose oxidation rate showed an increase in the lower significantly antagonized by incubation with an anti– millimolar concentration range, which does not necessar-

DIABETES, VOL. 53, APRIL 2004 1025 GK REGULATION BY PFK-2/FBPase-2

TABLE 1 Kinetic characteristics of GK in RINm5F-GK control cells and RINm5F-GK cells overexpressing islet or liver PFK-2/FBPase

Vmax (units/mg protein) S0.5 (mmol/l) nHill Control 8.6 Ϯ 0.6 3.2 Ϯ 0.3 1.4 Ϯ 0.2 RINm5F-GK cell clone Islet PFK-2/FBPase I4 19.1 Ϯ 0.9* 3.1 Ϯ 0.2 1.7 Ϯ 0.1 Liver PFK-2/FBPase L11 20.3 Ϯ 1.1* 3.7 Ϯ 0.3 2.6 Ϯ 0.3* Data are means Ϯ SE from four independent experiments. Glucokinase enzyme activity was measured in cell homogenates by a glucose-6-phosphate dehydrogenase–coupled spectrophotometric assay. Statistical analyses were performed with ANOVA followed by Bonferroni’s test for multiple comparisons. *P Ͻ 0.001 compared with RINm5F-GK control cells. ily reflect the situation in pancreatic islets and liver with a as deduced from the intracellular concentrations of F-2,6- predominant coupling of glucose metabolism by the GK P2. In RINm5F-GK cells, we observed low F-2,6-P2 levels enzyme. by overexpressing the liver isoform, high levels by over- To further evaluate the mechanisms by which PFK-2/ expressing the phosphatase-deficient liver isoform, and FBPase-2 activates GK the cAMP-sensitive liver isoform, a intermediate levels by overexpressing the islet isoform. liver mutant devoid of PKA phosphorylation sites and of Thus, it is unlikely that the activation of GK by PFK-2/ FBPase-2 activity as well as the islet/brain PFK-2/FBPase-2 FBPase-2 is due to generation of F-2,6-P2. Additionally, this isoform were stably expressed in RINm5F-GK cells. Nota- contention is supported by the fact that F-2,6-P2 had no bly, overexpression of the wild-type and mutant liver isoform significant effect on the kinetic characteristics of recom- as well as the islet/brain isoform of PFK-2/FBPase-2 re- binant GK protein and is therefore not an allosteric sulted in an increased GK enzyme activity (Fig. 3). Thus, effector of GK (S.B., unpublished data). PFK-2/FBPase-2 isoforms with different kinetic properties Notably, the lower intracellular levels of F-2,6-P2 in could activate the GK enzyme in insulin-producing cells. RINm5F-GK cells overexpressing the liver isoform of The bifunctional enzyme PFK-2/FBPase-2 controls the PFK-2/FBPase-2 apparently did not impair glucose metab- intracellular levels of F-2,6-P2. The physiological relevance olism. This was evident from the glucose oxidation rates in of F-2,6-P2 in glucose metabolism in pancreatic islets has the forskolin-treated liver PFK-2/FBPase-2 overexpressing been discussed previously and is controversial (49–51). RINm5F-GK cells, which were not significantly different However, there is a general consensus that glucose at from those of RINm5F-GK control cells (S.B., unpublished millimolar concentrations can increase F-2,6-P2 in pancre- data). Thus, the lower F-2,6-P2 levels in the L11 cell clone atic islets. This means that the kinase/bisphosphatase ratio apparently did not negatively affect glycolysis in insulin- of the bifunctional enzyme is dominated by the kinase producing cells. It should be emphasized that F-2,6-P2 is activity under physiological conditions. This raises the not an exclusive activator of PFK-1 in insulin-producing question of whether the activation of GK in PFK-2/FB- cells because long-chain acyl-CoA has been shown to be a Pase-2 overexpressing cells is related to the intracellular potent stimulator of this key glycolytic enzyme (52). Thus, level of F-2,6-P2. In the present study, overexpression of biofactors other than F-2,6-P2 may participate in the the different PFK-2/FBPase-2 isoforms in insulin-produc- regulation of the glycolytic pathway. ing cells resulted in variant kinase/bisphosphatase ratios Yeast two-hybrid studies clearly show that the GK protein interacts with the phosphatase domain of the PFK-2/FBPase-2 protein (23). The interaction with the FBPase-2 domain is a prerequisite for the activation of GK because this process could be efficiently blocked by an antibody raised against the FBPase-2 domain of the enzyme (Fig. 8). Our data indicate that the conformation of the whole bifunctional PFK-2/FBPase-2 enzyme plays an important role in the activation of GK. Forskolin, which shifted the activity ratio of the liver PFK-2/FBPase-2 isoform toward the FBPase-2 activity and modulated the conformation of the PFK-2/FBPase-2 homodimer (29), counteracted the activation of GK (Fig. 5). As expected, the effect of forskolin was evident only in RINm5F-GK cells overexpressing the cAMP-sensitive liver isoform of FIG. 8. Effect of anti–PFK-2/FBPase-2 antibody on GK enzyme activity in RINm5F-GK-PFK2 cells. Measurements were performed in RINm5F- PFK-2/FBPase-2 but not in cells overexpressing the islet/ GK–PFK-2/FBPase-2 islet clone I4, RINm5F-GK–PFK-2/FBPase-2 liver brain isoform or the mutant liver isoform, which lack the clone L11, and RINm5F-GK–PFK-2/FBPase-2 liver mutant clone LM12, phosphorylation site for cAMP-dependent protein kinase A compared with RINm5F-GK control cells. Cells were grown overnight at 10 mmol/l glucose. Thereafter, cells were homogenized by sonication (29). and incubated for1hinthepresence of FBPase-2 antibody (1:1,000). The prevention of GK activation by forskolin provides Enzyme activities were measured spectrophotometrically. Data are expressed as the percentage of enzyme activity measured in evidence that a high FBPase-2 activity of the phosphory- RINm5F-GK control cells in the absence of FBPase-2 antibody. lated liver PFK-2/FBPase-2 enzyme may significantly affect -Means ؎ SE from four to seven individual experiments are shown. binding to the GK protein through an unfavorable confor ***P < 0.001 compared with enzyme activity in untreated control cells; ###P < 0.001 compared with enzyme activity in the absence of FBPase-2 mation of the PFK-2/FBPase-2 protein. Thus, the cellular antibody (ANOVA/Bonferroni’s test). stoichometry between the phosphorylated and dephos-

1026 DIABETES, VOL. 53, APRIL 2004 L. MASSA AND ASSOCIATES phorylated PFK-2/FBPase-2 enzyme may be crucial for the demonstrated in insulin-producing cells. The activation of activation of GK by the liver isoform. The fructose-2,6- GK by PFK-2/FBPase-2 could also be demonstrated with bisphophate levels in RINm5F-GK cells overexpressing recombinant proteins in a reconstituted in vitro system liver PFK-2/FBPase-2 are lower than those of control cells, indicating that an interaction of the two proteins is suffi- providing evidence that PFK-2/FBPase-2 is predominantly cient to increase GK activity (S.B., unpublished data). The in the phosphorylated state. However, one has to take into regulatory principle of the GK interaction with PFK-2/ account that the F-2,6-P2 levels are an indirect indicator of FBPase-2 may also be of relevance for GK activity regula- the ratio between the phosphorylated and dephosphory- tion in liver because both proteins are abundantly lated form of PFK-2/FBPase-2 and are not conclusive with expressed in hepatocytes. In liver, the binding of GK to respect to the absolute amounts of the enzyme in relation- PFK-2/FBPase-2 has to be integrated into the concept of ship to those of the GK protein. In RINm5F-GK cells GK regulation through transcriptional activation of the GK overexpressing high levels of liver PFK-2/FBPase-2, the gene (4) and posttranslational modulation by the liver GK remaining portion of dephosphorylated enzyme may there- regulatory protein (26,60). An increase of the blood glu- fore be sufficient to activate the GK enzyme despite a cose concentration stimulates GK gene and protein ex- predominant phosphorylated form of PFK-2/FBPase-2. pression via insulin and/or high F-2,6-P2 concentrations This contention is supported by the fact that forskolin and the translocation of GK from the nucleus to the cytoplasm. At this point, the interaction of GK with significantly reduced the F-2,6-P2 levels, thereby shifting the ratio further toward the phosphorylated form of liver PFK-2/FBPase-2 may contribute to the increase of the GK PFK-2/FBPase-2. activity. Furthermore, the deactivation of GK by PKA in It is not clear at the moment if the PFK-2 domain of the the liver is consistent with the promotion of gluconeogen- PFK-2/FBPase-2 is required for the activation of the GK esis by glucagon, which proceeds via cAMP. In pancreatic ␤-cells, the GK gene is physiologically not enzyme. Mutation studies on recombinant liver PFK-2/ regulated by hormones on the level of enzyme protein, which FBPase-2 protein indicate that the phosphorylation of the remains more or less unaffected by the nutritional status enzyme by a PKA may induce conformational changes (13,61). A rationale for the stable levels of GK in pancreatic both at the NH - and COOH-termini of the protein, which 2 ␤-cells may be the indispensable glucose sensor function that are critical for the change of the kinase/bisphosphatase does not allow drastic changes of protein expression and activity ratio (29,53,54). enzyme activity as they occur in the liver in dependence upon The binding to PFK-2/FBPase-2 may stabilize a specific the induction by insulin and the repression by glucagon (7). conformation of the GK enzyme, resulting in a higher Thus, posttranslational enzyme regulation is crucial to main- catalytic activity. Another feasible explanation for the tain the glucose sensor function of GK in ␤-cells. Moreover, activation of GK could be a protection against intramolec- the tight coupling of the GK activity to insulin release via ular sulhydryl group oxidation within the enzyme protein. ATP/ADP suggests that the metabolic fluxes from glucose-6- In contrast to the high-affinity hexokinase isoenzymes phosphate through glycolysis, the tricarboxylic acid cycle, (types I–III), the GK protein is highly susceptible to and electron transport are all coordinated. sulhydryl group oxidation, although mammalian hexoki- The interaction of GK with the bifunctional enzyme nases share the same spatial cysteine pattern (17,18). This PFK-2/FBPase-2 is an entirely novel element in the post- GK sulfhydryl group oxidation occurs constitutively in an translational regulation of GK. There is evidence that the environment that does not provide protection, e.g., by thiol GK enzyme exists in two different activity states: 1)a reagents (16,17). freely diffusible state corresponding to a high intrinsic Importantly, PFK-2/FBPase-2 overexpression amplified activity through interaction with activating or stabilizing the stimulatory effect of glucose on the GK enzyme activity proteins such as the PFK-2/FBPase-2 and 2) a matrix- in RINm5F-GK cells (Fig. 6) and significantly increased the bound state to inhibitory proteins (13) and insulin granules Vmax of GK activity (Fig. 7, Table 1). Because the interac- (20,21,62) corresponding to a low intrinsic activity. Glu- tion did not affect the S0.5 of GK, the glucose sensor cose is the key molecule, which shifts the GK enzyme from function of the enzyme remains in the physiological con- a low activity state to a high activity state by a mechanism centration range. Thus, PFK-2/FBPase-2 acts as an ampli- that is as yet not completely understood on the molecular fier of glucose as the key regulator of GK activity on the level. However, there is evidence from kinetic data (14,63) posttranslational level (9–13,55). Glucose may act in a and recent studies with fluorescently labeled GK protein dual way, inducing a release of the GK protein from matrix (21) that glucose induces conformational changes, which proteins or secretory granules and, through a slow transi- are apparently responsible for the shift toward a high- tion, increasing the intrinsic activity by conformational activity state. Because chemical activators of GK are changes induced by the substrate glucose (13–15,21). under development as a new promising class of antidia- The posttranslational regulation of GK enzyme activity betic drugs (24), the knowledge of the GK-PFK-2/FBPase-2 by interaction with PFK-2/FBPase-2 in insulin-producing interaction may help to find the rationale for the physio- cells must be distinguished from the activation of GK logical upregulation of GK and new perspective therapeu- mRNA and protein expression by F-2,6-P2 in liver (56,57). tic strategies to activate or preserve the glucose sensor The ability of elevated F-2,6-P2 concentrations to upregu- function of GK in type 2 diabetes. late GK gene expression in the absence of insulin (56–58) is a unique characteristic of the hepatic GK regulating system due to tissue-specific promoters (4,59). ACKNOWLEDGMENTS In the present study, the activation of GK enzyme This work was supported by a grant from the Ministry of activity by interaction with PFK-2/FBPase-2 has been Science and Culture of Lower Saxony (to L.M.) and the

DIABETES, VOL. 53, APRIL 2004 1027 GK REGULATION BY PFK-2/FBPase-2

DFG (German Research Foundation) (to S.L.). A.J.L. was glucokinase-binding protein epitopes by a phage-displayed peptide library: supported by National Institutes of Health Grant DK- identification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase as a novel interaction partner. J Biol Chem 276:43915–43923, 2001 38354. 24. Grimsby J, Sarabu R, Corbett WL, Haynes NE, Bizzarro FT, Coffey JW, The authors acknowledge the excellent technical assis- Guertin KR, Hilliard DW, Kester RF, Mahaney PE, Marcus L, Qi L, Spence tance of M. Boeger and B. Lueken. The authors are grateful CL, Tengi J, Magnuson MA, Chu CA, Dvorozniak MT, Matschinsky FM, to Dr. B. Vogelstein (Baltimore, MD) for providing the Grippo JF: Allosteric activators of glucokinase: potential role in diabetes vectors of the adenoviral expression system and to Dr. M. therapy. Science 301:370–373, 2003 Asfari (Paris, France) for providing the INS1 cells. 25. Van Schaftingen E: Short-term regulation of glucokinase. Diabetologia 37 (Suppl. 2):S43–S47, 1994 26. Van Schaftingen E, Detheux M, Veiga da Cunha M: Short-term control of REFERENCES glucokinase activity: role of a regulatory protein. FASEB J 8:414–419, 1994 27. Claus TH, El-Maghrabi MR, Regen DM, Stewart HB, McGrane M, Kountz 1. Meglasson MD, Matschinsky FM: New perspectives on pancreatic islet PD, Nyfeler F, Pilkis J, Pilkis SJ: The role of fructose 2,6-bisphosphate in glucokinase. Am J Physiol 246:E1–E13, 1984 the regulation of carbohydrate metabolism. Curr Top Cell Regul 23:57–86, 2. Lenzen S, Panten U: Signal recognition by pancreatic B-cells. Biochem 1984 Pharmacol 37:371–378, 1988 28. Pilkis SJ, Claus TH, Kurland IJ, Lange AJ: 6-Phosphofructo-2-kinase/ 3. Magnuson MA: Glucokinase gene structure: functional implications of fructose-2,6-bisphosphatase: a metabolic signaling enzyme. Annu Rev molecular genetic studies. Diabetes 39:523–527, 1990 4. Iynedjian PB: Mammalian glucokinase and its gene. Biochem J 293:1–13, Biochem 64:799–835, 1995 1993 29. Okar DA, Manzano A, Navarro-Sabate A, Riera L, Bartrons R, Lange AJ: 5. Matschinsky F, Liang Y, Kesavan P, Wang L, Froguel P, Velho G, Cohen D, PFK-2/FBPase-2: maker and breaker of the essential biofactor fructose- Permutt MA, Tanizawa Y, Jetton TL, Niswender K, Magnuson MA: Glucoki- 2,6-bisphosphate. Trends Biochem Sci 26:30–35, 2001 nase as pancreatic beta cell glucose sensor and diabetes gene. J Clin 30. Gazdar A, Chick W, Oie H, Sims H, King D, Weir G, Lauris V: Continuous, Invest 92:2092–2098, 1993 clonal, insulin- and somatostatin-secreting cell lines established from a 6. Matschinsky FM: Banting Lecture 1995: A lesson in metabolic regulation transplantable rat islet cell tumor. Proc Natl Acad SciUSA77:3519–3523, inspired by the glucokinase glucose sensor paradigm. Diabetes 45:223–241, 1980 1996 31. Bhathena S, Oie H, Gazdar A, Voyles N, Wilkins S, Recant L: Insulin, 7. Matschinsky FM: Regulation of pancreatic beta-cell glucokinase: from glucagon, and somatostatin receptors on cultured cells and clones from rat basics to therapeutics. Diabetes 51 (Suppl. 3):S394–S404, 2002 islet cell tumor. Diabetes 31:521–531, 1982 8. Chen C, Bumbalo L, Leahy JL: Increased catalytic activity of glucokinase in 32. Koranyi LI, Tanizawa Y, Welling CM, Rabin DU, Permutt MA: Human islet isolated islets from hyperinsulinemic rats. Diabetes 43:684–689, 1994 glucokinase gene: isolation and sequence analysis of full-length cDNA. 9. Chen C, Hosokawa H, Bumbalo LM, Leahy JL: Regulatory effects of glucose Diabetes 41:807–811, 1992 on the catalytic activity and cellular content of glucokinase in the 33. Tiedge M, Elsner M, McClenaghan NH, Hedrich HJ, Grube D, Klempnauer pancreatic beta cell: study using cultured rat islets. J Clin Invest 94:1616– J, Lenzen S: Engineering a glucose responsive surrogate cell for insulin 1620, 1994 replacement therapy of experimental insulin-dependent diabetes. Hum 10. Liang Y, Jetton TL, Zimmerman EC, Najafi H, Berner DK, Matschinsky FM, Genet Ther 11:403–414, 2000 Magnuson MA: Effects of glucose on insulin secretion, glucokinase activ- 34. Asfari M, Janjic D, Meda P, Li G, Halban PA, Wollheim CB: Establishment ity, and transgene expression in transgenic mouse islets containing an of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. upstream glucokinase promoter-human growth hormone fusion gene. Endocrinology 130:167–178, 1992 Diabetes 43:1138–1145, 1994 35. Darville MI, Crepin KM, Vandekerckhove J, Van Damme J, Octave JN, 11. Liang Y, Najafi H, Matschinsky FM: Glucose regulates glucokinase activity Rider MH, Marchand MJ, Hue L, Rousseau GG: Complete nucleotide in cultured islets from rat pancreas. J Biol Chem 265:16863–16866, 1990 sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6- 12. Liang Y, Najafi H, Smith RM, Zimmerman EC, Magnuson MA, Tal M, bisphosphatase derived from a cDNA clone. FEBS Lett 224:317–321, 1987 Matschinsky FM: Concordant glucose induction of glucokinase, glucose 36. Argaud D, Lange AJ, Becker TC, Okar DA, el-Maghrabi MR, Newgard CB, usage, and glucose-stimulated insulin release in pancreatic islets main- Pilkis SJ: Adenovirus-mediated overexpression of liver 6-phosphofructo-2- tained in organ culture. Diabetes 41:792–806, 1992 kinase/fructose-2,6-bisphosphatase in gluconeogenic rat hepatoma cells:

13. Tiedge M, Steffeck H, Elsner M, Lenzen S: Metabolic regulation, activity paradoxical effect on Fru-2,6-P2 levels. J Biol Chem 270:24229–24236, 1995 state, and intracellular binding of glucokinase in insulin-secreting cells. 37. Argaud D, Kirby T, Newgard C, Lange A: Stimulation of glucose-6- Diabetes 48:514–523, 1999 phosphatase gene expression by glucose and fructose-2,6-bisphosphate. 14. Neet KE, Keenan RP, Tippett PS: Observation of a kinetic slow transition J Biol Chem 272:12854–12861, 1997 in monomeric glucokinase. Biochemistry 29:770–777, 1990 38. Watanabe F, Sakai A, Furuya E, Uyeda K: Molecular cloning and tissue 15. Neet KE: Cooperativity in enzyme function: equilibrium and kinetic specific expression of fructose 6-phosphate,2-kinase:fructose 2,6-bisphos- aspects. Methods Enzymol 249:519–567, 1995 phatase of rat brain. Biochem Biophys Res Commun 198:335–340, 1994 16. Tippett PS, Neet KE: Interconversions between different sulfhydryl-related 39. Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory kinetic states in glucokinase. Arch Biochem Biophys 222:285–298, 1983 Manual. New York, Cold Spring Harbor Laboratory Press, 1989 17. Tiedge M, Richter T, Lenzen S: Importance of cysteine residues for the 40. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B: A simplified stability and catalytic activity of human pancreatic beta cell glucokinase. system for generating recombinant adenoviruses. Proc Natl Acad Sci U S Arch Biochem Biophys 375:251–260, 2000 A 95:2509–2514, 1998 18. Tiedge M, Krug U, Lenzen S: Modulation of human glucokinase intrinsic 41. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid activity by SH reagents mirrors post-translational regulation of enzyme guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem activity. Biochim Biophys Acta 1337:175–190, 1997 162:156–159, 1987 19. Noma Y, Bonner Weir S, Latimer JB, Davalli AM, Weir GC: Translocation 42. Kurland IJ, Li L, Lange AJ, Correia JJ, el-Maghrabi MR, Pilkis SJ: Regula- of glucokinase in pancreatic beta-cells during acute and chronic hypergly- tion of rat 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: role of cemia. Endocrinology 137:1485–1491, 1996 the NH2-terminal region. J Biol Chem 268:14056–14064, 1993 20. Toyoda Y, Yoshie S, Shironoguchi H, Miwa I: Glucokinase is concentrated 43. Lemamy GJ, Roger P, Mani JC, Robert M, Rochefort H, Brouillet JP: in insulin-secretory granules of pancreatic B-cells. Histochem Cell Biol High-affinity antibodies from hen’s-egg yolks against human mannose-6- 112:35–40, 1999 phosphate/insulin-like growth-factor-II receptor (M6P/IGFII-R): character- 21. Rizzo MA, Magnuson MA, Drain PF, Piston DW: A functional link between ization and potential use in clinical cancer studies. Int J Cancer 80:896– glucokinase binding to insulin granules and conformational alterations in 902, 1999 response to glucose and insulin. J Biol Chem 277:34168–34175, 2002 44. Lenzen S, Tiedge M, Panten U: Glucokinase in pancreatic B-cells and its 22. Shiraishi A, Yamada Y, Tsuura Y, Fijimoto S, Tsukiyama K, Mukai E, inhibition by alloxan. Acta Endocrinol (Copenh) 115:21–29, 1987 Toyoda Y, Miwa I, Seino Y: A novel glucokinase regulator in pancreatic 45. Cornish-Bowden A: Fundamentals of Enzyme Kinetics. London, Butter- beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts worth, 1979 with glucokinase and augments its activity. J Biol Chem 276:2325–2328, 46. Cascante M, Centelles JJ, Agius L: Use of alpha-toxin from Staphylococcus 2001 aureus to test for channeling of intermediates of glycolysis between 23. Baltrusch S, Lenzen S, Okar DA, Lange AJ, Tiedge M: Characterization of glucokinase and aldolase in hepatocytes. Biochem J 352:899–905, 2000

1028 DIABETES, VOL. 53, APRIL 2004 L. MASSA AND ASSOCIATES

47. Van Schaftingen E, Lederer B, Bartrons R, Hers HG: A kinetic study of hypertensive rats: augmented enzymatic activity of glucokinase in beta- pyrophosphate: fructose-6-phosphate phosphotransferase from potato tu- cells. J Clin Invest 94:399–404, 1994 bers: application to a microassay of fructose 2,6-bisphosphate. Eur J Bio- 56. Wu C, Okar DA, Newgard CB, Lange AJ: Overexpression of 6-phospho- chem 129:191–195, 1982 fructo-2-kinase/fructose-2, 6-bisphosphatase in mouse liver lowers blood 48. Lenzen S, Panten U: 2-Oxocarboxylic acids and function of pancreatic glucose by suppressing hepatic glucose production. J Clin Invest 107:91– islets in obese-hyperglycaemic mice. Biochem J 186:135–144, 1980 98, 2001 49. Malaisse WJ, Malaisse-Lagae F, Sener A: Glucose-induced accumulation of 57. Wu C, Okar DA, Newgard CB, Lange AJ: Increasing fructose 2,6-bisphos- fructose-2,6-bisphosphate in pancreatic islets. Diabetes 31:90–93, 1982 phate overcomes hepatic insulin resistance of type 2 diabetes. Am J Physiol Endocrinol Metab 282:E38–E45, 2002 50. Sener A, Van Schaftingen E, Van de Winkel M, Pipeleers DG, Malaisse- 58. Choi IY, Wu C, Okar DA, Lange AJ, Gruetter R: Elucidation of the role of Lagae F, Malaisse WJ, Hers HG: Effects of glucose and glucagon on the fructose 2,6-bisphosphate in the regulation of glucose fluxes in mice using fructose 2,6-bisphosphate content of pancreatic islets and purified pancre- in vivo (13)C NMR measurements of hepatic carbohydrate metabolism. atic B-cells: a comparison with isolated hepatocytes. Biochem J 221:759– Eur J Biochem 269:4418–4426, 2002 764, 1984 59. Magnuson MA, Niswender KD, Pettepher CC: Glucokinase gene expres- 51. Burch PT, Berner DK, Najafi H, Meglasson MD, Matschinsky FM: Regula- sion and regulation. In Molecular Biology of Diabetes, Part I. Draznin B, tory role of fructose-2,6-bisphosphate in pancreatic islet glucose metabo- LeRoith D, Eds. Totowa, Humana Press, 1994, p. 155–174 lism remains unsettled. Diabetes 34:1014–1018, 1985 60. Agius L, Peak M: Intracellular binding of glucokinase in hepatocytes and 52. Liu YQ, Tornheim K, Leahy JL: Fatty acid-induced beta cell hypersensitiv- translocation by glucose, fructose and insulin. Biochem J 296:785–796, ity to glucose: increased phosphofructokinase activity and lowered glu- 1993 cose-6-phosphate content. J Clin Invest 101:1870–1875, 1998 61. Iynedjian PB, Pilot PR, Nouspikel T, Milburn JL, Quaade C, Hughes S, Ucla 53. Kurland IJ, el-Maghrabi MR, Correia JJ, Pilkis SJ: Rat liver 6-phospho- C, Newgard CB: Differential expression and regulation of the glucokinase fructo-2-kinase/fructose-2,6-bisphosphatase: properties of phospho- and gene in liver and islets of Langerhans. Proc Natl Acad SciUSA dephospho- forms and of two mutants in which Ser32 has been changed by 86:7838–7842, 1989 site-directed mutagenesis. J Biol Chem 267:4416–4423, 1992 62. Toyoda Y, Yoshie S, Fujita T, Ito Y, Nonogaki T, Miwa I: Glucokinase is 54. Lee YH, Li Y, Uyeda K, Hasemann CA: Tissue-specific structure/function located in secretory granules of pancreatic D-cells. FEBS Lett 415:281–284, differentiation of the liver isoform of 6-phosphofructo-2-kinase/fructose- 1997 2,6-bisphosphatase. J Biol Chem 278:523–530, 2003 63. Lin SX, Neet KE: Demonstration of a slow conformational change in liver 55. Chen C, Hosokawa H, Bumbalo LM, Leahy JL: Mechanism of compensa- glucokinase by fluorescence spectroscopy. J Biol Chem 265:9670–9675, tory hyperinsulinemia in normoglycemic insulin-resistant spontaneously 1990

DIABETES, VOL. 53, APRIL 2004 1029