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Endocrinology, doi:10.1210/en.2006-0694
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Endocrinology Vol. 147, No. 12 5768-5776
Copyright © 2006 by The Endocrine Society

Improved Metabolic Stimulus for Glucose-Induced Insulin Secretion through GK and PFK-2/FBPase-2 Coexpression in Insulin-Producing RINm5F Cells

Simone Baltrusch, Sara Langer, Laura Massa, Markus Tiedge and Sigurd Lenzen

Institute of Clinical Biochemistry (S.B., S.La., L.M., M.T., S.Le.), Hannover Medical School, 30623 Hannover, Germany; and Institute of Medical Biochemistry and Molecular Biology (M.T.), University of Rostock, 18057 Rostock, Germany

Address all correspondence and requests for reprints to: Dr. Simone Baltrusch, Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany. E-mail: baltrusch.simone{at}mh-hannover.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The glucose sensor enzyme glucokinase plays a pivotal role in the regulation of glucose-induced insulin secretion in pancreatic ß-cells. Activation of glucokinase represents a promising concept for the treatment of type 2 diabetes. Therefore, we analyzed the glucokinase activation through its physiological interaction partner, the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) and the resulting effect on glucose metabolism in insulin-producing cells. In RINm5F-GK-PFK-2/FBPase-2 cells stably overexpressing glucokinase plus islet PFK-2/FBPase-2, colocalization between both enzymes as well as elevation of glucokinase activity were significantly increased at a stimulatory glucose concentration of 10 mmol/liter. RINm5F-GK-PFK-2/FBPase-2 cells showed under this culture condition a significant increase in glucose utilization and in the ATP/ADP ratio compared with RINm5F-GK cells, which only overexpress glucokinase. Also glucose-induced insulin secretion was elevated in RINm5F-GK-PFK-2/FBPase-2 cells in comparison to RINm5F-GK cells. Furthermore, pyruvate accumulation and lactate production in RINm5F-GK-PFK-2/FBPase-2 cells were significantly lower at both 10 and 30 mmol/liter glucose than in RINm5F-GK and RINm5F cells. The significant improvement of glucose metabolism after PFK-2/FBPase-2 overexpression is apparently not exclusively the result of high glucokinase enzyme activity. Stabilization of the closed glucokinase conformation by PFK-2/FBPase-2 may not only activate the enzyme but also improve metabolic channeling in ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GLUCOSE PHOSPHORYLATING enzyme glucokinase, in pancreatic ß-cells and liver, but also in enteroendocrine cells, pituitary cells, and neurons, has the glucose sensor function due to its cooperative kinetics and high control strength in the regulation of glycolysis (1, 2, 3, 4, 5, 6, 7, 8). In ß-cells, glucokinase catalyzes the rate-limiting step of glucose-induced insulin secretion (6). Therefore, the elucidation of the mechanisms underlying glucokinase regulation in ß-cells on the gene transcription level and especially by posttranslational mechanisms has gained particular interest (8). The glucose-dependent glucokinase conformational change between the closed and super-open form plays a fundamental role in the glucokinase activity modulation (9). Furthermore, glucokinase binding partners like ß-cell matrix proteins and insulin secretory granules may affect cytoplasmic compartmentation of glucokinase (10, 11, 12, 13, 14). Interestingly, recently discovered small chemical compounds activate glucokinase by binding to an allosteric site of the enzyme protein. These so-called glucokinase activators have the ability to increase glucose-stimulated insulin release and thereby provide the rationale for a promising new concept of type 2 diabetes pharmacotherapy (9, 15, 16, 17). Glucokinase activation is also physiologically mediated by interaction with the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) (18, 19). ß-Cells express the brain isoform of PFK-2/FBPase-2, which is not regulated by phosphorylation and dephosphorylation (20). In recent studies, we could show that overexpression of PFK-2/FBPase-2 in insulin-producing cells results in a significant increase of glucokinase enzyme activity and glucose oxidation, indicating a beneficial effect on glucose-induced insulin secretion (18).

Therefore, the aim of our present study was to characterize key features of glucose metabolism in insulin-producing RINm5F cells overexpressing glucokinase plus islet PFK-2/FBPase-2 (RINm5F-GK-PFK-2/FBPase-2 cells) or glucokinase alone (RINm5F-GK cells). The results show that activated glucokinase plays a central role in the flux control of glycolysis and improves oxidative glucose metabolism and glucose-induced insulin secretion with a concomitant decrease in pyruvate and lactate accumulation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The enhanced chemiluminescence detection system, autoradiography films, and radiochemicals were from Amersham Pharmacia Biotech (Freiburg, Germany). Forskolin was from ICN Biomedicals (Irvine, CA). All reagents of analytical grade were from Merck (Darmstadt, Germany). All tissue culture equipment was from Invitrogen (Karlsruhe, Germany).

RINm5F cell culture
Insulin-producing RINm5F cells overexpressing glucokinase (RINm5F-GK cells) were generated by stable transfection of the human ß-cell glucokinase cDNA as described previously (11, 21). RINm5F-GK cells overexpressing PFK-2/FBPase-2 were generated by a second stable transfection of the cDNA for rat liver (RINm5F-GK-PFK-2/FBPase-2 L 11), for the rat liver S32A/H258A double mutant (RINm5F-GK-PFK-2/FBPase-2 LM 12), or for rat islets (RINm5F-GK-PFK-2/FBPase-2 I 4) as described previously (18). Cells were grown in RPMI 1640 medium supplemented with 10 mmol/liter glucose, 10% (vol/vol) fetal calf serum (FCS), penicillin, and streptomycin in a humidified atmosphere at 37 C and 5% CO2. The medium for RINm5F-GK cells was additionally supplemented with 250 µg/ml G418, and the medium for RINm5F-GK-PFK-2/FBPase-2 cells with 250 µg/ml G418 and 250 µg/ml Zeocin. For gene silencing, RINm5F-GK-PFK-FBPase-2 islet (I 4) cells were seeded in six-well microplates at a density of 1.5 x 105. On the next day, cells were transfected with either 1.5 µg islet PFK-2/FBPase-2 cDNA-specific small interfering RNA (siRNA) or control siRNA and 4 µl jetSI-ENDO (Polyplus, Illkirch, France) for 4 h in RPMI 1640 medium in the absence of FCS according to the manufacturer’s instructions. Thereafter, cells were incubated for 48 h in RPMI 1640 medium supplemented with 10 mmol/liter glucose and 5% FCS. The islet PFK-2/FBPase-2 cDNA specific siRNA duplex was designed against a target sequence of the islet PFK-2/FBPase-2 cDNA (CCA GAG TAA GAT TGT CTA CTA, GenBank accession no. S67900) by the HiPerformance Design Algorithm licensed from Novartis AG (QIAGEN, Hilden, Germany).

Glucose phosphorylation and glucokinase enzyme activity
Glucose phosphorylating activity was measured at various glucose concentrations (1, 1.56, 3.12, 6.25, 12.5, and 25 mmol/liter) in soluble cellular fractions of RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells by an enzyme-coupled photometric assay as described previously (22). One unit of enzyme activity was defined as 1 µmol glucose-6-phosphate formed from glucose and ATP per minute at 37 C. Enzyme activity was expressed as units per mg cellular protein. Glucokinase activity was determined by subtracting the hexokinase activity measured at 1 mmol/liter glucose from the activity measured at 100 mmol/liter glucose.

Western blot analyses
Glucokinase and PFK-2/FBPase-2 Western blot analyses and glucokinase activity measurements were performed from identical samples to allow a direct comparison between protein expression and enzyme activity. The cells were homogenized in PBS (pH 7.4), and insoluble material was pelleted by centrifugation. The protein concentration was quantified by a Bio-Rad protein assay. Forty micrograms of cellular protein were fractionated by reducing 10% SDS-PAGE and electroblotted to polyvinylidine difluoride membranes. The membranes were stained by Ponceau to verify the transfer of comparable amounts of cellular protein. Nonspecific binding sites of the membranes were blocked by nonfat dry milk overnight at 4 C. Glucokinase and PFK-2/FBPase-2 immunodetection was performed as described (11, 18).

Immunostaining
Cells were seeded on glass cover slips and grown for 24 h in medium with 10 mmol/liter glucose. Thereafter, cells were incubated for the next 24 h in medium with either 3 or 10 mmol/liter glucose. Finally, cells were washed twice with PBS (pH 7.4), fixed with ice-cold acetone for 5 min, and treated for 20 min with 0.2% Triton X-100 and 1% BSA in PBS. Cover slips were washed three times with PBS and incubated for 1 h with the glucokinase antibody (11) and the FBPase-2 antibody (18), both diluted 1:1000 in PBS supplemented with 0.1% Triton X-100 and 1% BSA. Thereafter, cover slips were washed three times with PBS and incubated for 1 h with fluorescein isothiocyanate (FITC) donkey antirabbit antibody for glucokinase and Texas Red (TxRed) donkey antichicken antibody for FBPase-2, both diluted 1:200 in PBS supplemented with 0.1% Triton X-100 and 1% BSA. Finally, cover slips were washed three times with PBS and mounted with ProLong antifade reagent in mounting medium (Molecular Probes Invitrogen Detection Technologies, Eugene, OR) onto slides. Images were taken with an Olympus IX81/cell system as described previously (23). S 492/18 and S 572/23 excitation filters were used for FITC and TxRed, respectively, and a DAPI/FITC/TxRed triple band beam splitter and emitter (AHF Analysentechnik, Tübingen, Germany). Colocalization was calculated with Image J 1.32 (W. Rasband, National Institutes of Health) using the plug-in module Colocalization Finder (C. Laummonerie, Strasbourg, France).

Measurements of fructose-2,6-bisphosphate (F-2,6-P2)
RINm5F, RINm5F-GK, RINm5F-GK-PFK-2/FBPase-2 (L 11), RINm5F-GK-PFK-2/FBPase-2 (LM 12), and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells were grown for 24 h in medium with 10 mmol/liter glucose. Thereafter, cells were incubated for 2 h in the presence of 10 µM forskolin. Finally, cells were permeabilized by 10 µg/ml {alpha}-toxin from Staphylococcus aureus (Sigma, Taufkirchen, Germany) as described (18). F-2,6-P2 was determined in the supernatant by an enzyme-coupled method using pyrophosphate-dependent fructose-6-phosphate kinase (Sigma).

Glucose metabolism
The glucose utilization rate was assessed as the production of 3H2O from D-[5-3H]glucose. Glucose metabolism was measured as described (24, 25) in batches of 5 x 105 cells over a 1-h incubation at 37 C in 40 µl of Krebs-Ringer buffer containing various glucose concentrations (0.5, 1, 2, 5, and 10 mmol/liter). Total radioactivity added to the cells was 0.4 µCi/ml. Cellular metabolism was arrested by the addition of 50 µl of 0.2 M HCl, and the produced 3H2O was captured by 500 µl H2O. After overnight incubation at 37 C, wells containing cells were removed and the scintillation liquid was added. The radioactivity was counted in a liquid-scintillation spectrometer.

Determination of ATP and ADP
Cells were seeded in six-well microplates at a density of 5 x 105 cells and grown for 24 h in medium with 10 mmol/liter glucose. Thereafter, cells were incubated for the next 48 h in medium with either 3 or 10 mmol/liter glucose. Analysis of ATP was performed with the ATPlite Detection Assay System (PerkinElmer Life Sciences, Zaventem, Belgium) according to the manufacturer’s instructions. This system is based on the production of light caused by the reaction of ATP with added luciferase and D-luciferin (26). The attached mammalian cell lysis solution releases the adenine nucleotides and inactivates endogenous ATP degrading enzymes. For determining the ADP content, the sum of the ATP content and the ADP content was measured by conversion of ADP to ATP with pyruvate kinase and phosphoenolpyruvate for 15 min. Light emission was recorded as a 1-sec integral in a microplate using a Victor2 luminometer (Wallac, Freiburg, Germany) and the protein concentration was quantified by a Bio-Rad (Hercules, CA) protein assay. ATP/ADP values were calculated in relation to ATP standard values and protein content.

Measurements of pyruvate and lactate
Cells were grown for 72 h in medium with 10 mmol/liter glucose or for 24 h in medium with 10 mmol/liter glucose and for the next 48 h in medium with 30 mmol/liter glucose. Thereafter, batches of 2 x 106 cells were incubated for 1 h at 37 C and 300 rpm in 1 ml Krebs-Ringer buffer in the presence of various glucose concentrations (0.5, 1, 2, 5, and 10 mmol/liter). The cellular solution was gently centrifuged to remove the cells, and the supernatant was heated to 95 C for a period of 5 min to denature proteins. Thereafter, pyruvate production was determined by measuring the oxidation of reduced nicotinamide adenine dinucleotide in the presence of lactate dehydrogenase, and lactate production was determined by measuring the reduction of nicotinamide adenine dinucleotide in the presence of lactate dehydrogenase, glutamate, and glutamate-pyruvate transaminase as described (27, 28).

Insulin secretion and insulin content
Cells were seeded in six-well microplates at a density of 5 x 105 cells and grown for 48 h in medium with 10 mmol/liter glucose. Cells were then washed twice with medium without glucose and preincubated for 1 h in medium without glucose. Insulin secretion during a 1-h incubation period was measured in Krebs-Ringer buffer without glucose or in the presence of various glucose concentrations (1, 3, 5, 10, and 25 mmol/liter) or in the presence of 25 mmol/liter KCl. Thereafter, for measurements of insulin secretion, the incubation buffer was carefully removed and gently centrifuged to delete detached cells. Insulin content was measured in the soluble fraction of the homogenized cells. Insulin was determined by RIA using a rat insulin standard.

MTT cell viability assay
RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells were seeded in 96-well microplates at a density of 5,000 cells per well in 100-µl medium with 10 mmol/liter glucose and cultured for 24 h. Thereafter, cells were incubated for the next 48 h in medium with 3, 10, or 30 mmol/liter glucose. The cell viability was determined using a microtiter plate-based MTT assay as described (29). The decrease of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) activity is a reliable metabolically based test for quantification of cell viability (30).

Statistical analyses
The data are expressed as means ± SEM. Statistical analyses were performed by ANOVA followed by Bonferroni’s test for multiple comparison or Student’s t test using the Prism analysis program (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Relationship between glucokinase activation and PFK-2/FBPase-2 enzyme level
Stable overexpression of the islet PFK-2/FBPase-2 isoform in RINm5F-GK cells resulted in a significant increase in the PFK-2/FBPase-2 protein level to 364% and in a significant increase in glucokinase enzyme activity to 178%, whereas glucokinase protein expression levels were not affected (18). The S0.5 values for glucose phosphorylation were 3.2 mmol/liter in RINm5F-GK cells and 3.1 mmol/liter in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells, and were thus lower than in pancreatic islets probably due to the high activity levels of low-Km hexokinases (18). Although RINm5F cells showed no demonstrable increase in the phosphorylating activity in response to glucose, glucokinase overexpressing RINm5F-GK cells exhibited an increase in the phosphorylating activity at 3.12 mmol/liter and higher glucose concentrations (Fig. 1Go). Interestingly, only at these glucose concentrations did RINm5F-GK-PFK-2/FBPase-2 (I 4) show a higher phosphorylating activity than RINm5F-GK cells (Fig. 1Go). Thus, the increase of phosphorylating activity in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells compared with RINm5F-GK cells was apparently conferred by activation of the glucokinase enzyme and seems to be independent of the hexokinase enzyme. Furthermore, insulin-producing RINm5F-GK-PFK-2/FBPase-2 (I 4) cells were transfected with islet PFK-2/FBPase-2 cDNA-specific siRNA, which resulted in a reduction of the PFK-2/FBPase-2 protein level by 48% compared with cells transfected with control siRNA (Fig. 2AGo). The increase in glucokinase enzyme activity in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells to 178% could be significantly reduced to 133% by transfection with the islet PFK-2/FBPase-2 cDNA specific siRNA, but not with control siRNA (Fig. 2BGo). Glucokinase protein expression levels were not affected by down-regulation of PFK-2/FBPase-2 (Fig. 2CGo). Thus, glucokinase was activated by PFK-2/FBPase-2 on the posttranslational level.


Figure 1
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  		FIG. 1. Effects of glucokinase overexpression alone and glucokinase plus islet PFK-2/FBPase-2 co-overexpression in RINm5F-cells on the glucose phosphorylating activity. Cells were grown overnight at 10 mmol/liter glucose. Thereafter, RINm5F cells (white bars), RINm5F-GK cells (gray bars), and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells (black bars) were homogenized by sonication, and phosphorylating activities were determined spectrophotometrically in the presence of various glucose concentrations (1, 1.56, 3.12, 6.25, 12.5, and 25 mmol/liter). Shown are means ± SEM from four individual experiments. *, P < 0.05; **, P < 0.01 compared with RINm5F-GK cells (ANOVA/Bonferroni’s test).

 

Figure 2
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  		FIG. 2. Down-regulation of PFK-2/FBPase-2 in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells by siRNA. Cells were transfected with islet PFK-2/FBPase-2 cDNA-specific siRNA (black bar) or with control siRNA (white bar) for 4 h in RPMI 1640 medium in the absence of FCS. Thereafter, cells were incubated for 48 h in RPMI 1640 medium supplemented with 10 mmol/liter glucose and 5% FCS. For Western blot analyses, 40 µg cellular protein was analyzed per lane by immunoblotting using a specific antibody against FBPase-2 (A) or glucokinase (C). Shown are representative blots of four independent experiments. Glucokinase enzyme activities (B) were measured spectrophotometrically in cell extracts after sonication. Data are expressed as percentage of enzyme activity measured in RINm5F-GK control cells. Shown are means ± SEM from four individual experiments. **, P < 0.01 compared with cells transfected with control siRNA (Student’s t test).

 
F-2,6-P2 metabolism in RINm5F cells
Overexpression of the islet PFK-2/FBPase-2 isoform in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells did not affect the F-2,6-P2 concentration in comparison to RINm5F-GK cells. The protein kinase A activator forskolin provoked a nonsignificant decrease of the F-2,6-P2 concentration by 26% in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells, which was also observed in RINm5F-GK (23%) cells and untransfected RINm5F (24%) cells (Fig. 3Go). Thus, neither glucokinase nor the overexpression of the tissue-specific islet PFK-2/FBPase-2 isoform affected the regulation of the F-2,6-P2 concentration in insulin-producing cells.


Figure 3
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  		FIG. 3. Effects of forskolin on the F-2,6-P2 content in RINm5F, RINm5F-GK, RINm5F-GK-PFK-2/FBPase-2 (L 11), RINm5F-GK-PFK-2/FBPase-2 (LM 12), and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. Cells were grown for 24 h in medium with 10 mmol/liter glucose. Thereafter, cells were incubated for 2 h in the absence (white bars) or presence (black bars) of 10 µM forskolin. Finally, cells were permeabilized by 10 µg/ml {alpha}-toxin from S. aureus, and the F-2,6-P2 was determined spectrophotometrically. Shown are means ± SEM from four to seven individual experiments. *, P < 0.05 compared with the respective untreated cells; ##, P < 0.01 compared with RINm5F-GK cells (Student’s t test).

 
Although overexpression of the liver PFK-2/FBPase-2 isoform in RINm5F-GK-PFK-2/FBPase-2 (L11) cells significantly reduced the cellular F-2,6-P2 concentration by 66%, RINm5F-GK-PFK-2/FBPase-2 (LM 12) cells overexpressing a phosphorylation insensitive mutant showed a 53% higher concentration of F-2,6-P2 than RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. This indicates alterations in the F-2,6-P2 concentration due to expression of another PFK-2/FBPase-2 isoform in insulin-producing cells. Furthermore, in comparison with RINm5F-GK cells there was a comparable increase in the PFK-2/FBPase-2 protein level to 364% in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells and to 362% in RINm5F-GK-PFK-2/FBPase-2 (L11) cells, whereas in RINm5F-GK-PFK-2/FBPase-2 (LM 12) cells the liver mutant PFK-2/FBPase-2 protein level was elevated to 415%. Considering this somewhat higher expression level of the latter clone, the RINm5F-GK-PFK-2/FBPase-2 (LM 12) cells showed only a 38% higher concentration of F-2,6-P2 compared with RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. Forskolin significantly reduced the F-2,6-P2 concentration by 68% in cells overexpressing the liver PFK-2/FBPase-2 isoform, whereas RINm5F-GK-PFK-2/FBPase-2 (LM 12) cells showed only a decrease of the F-2,6-P2 concentration by 24% comparable to RINm5F-GK-PFK-2/FBPase-2 (I 4), RINm5F-GK, and RINm5F cells (Fig. 3Go). Thus, the low F-2,6-P2 concentration and the inhibitory effect of forskolin in RINm5F-GK-PFK-2/FBPase-2 (L 11) cells could be mediated through the regulatory phosphorylation site for protein kinase A in the liver isoform, which is not present in the PFK-2/FBPase-2 islet isoform.

Glucose-dependent interaction of glucokinase with islet PFK-2/FBPase-2
PFK-2/FBPase-2 and glucokinase immunofluorescence was detectable in the cytoplasm of RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. Colocalization between glucokinase and PFK-2/FBPase-2, indicating interaction between both proteins, was observed in RINm5F-GK and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells cultured at 10 mmol/liter glucose (data not shown). Interestingly, RINm5F-GK-PFK-2/FBPase-2 (I 4) cells showed less colocalization between glucokinase and PFK-2/FBPase-2 at 3 mmol/liter glucose (Fig. 4AGo) than at 10 mmol/liter glucose (Fig. 4BGo). Quantitative analyses revealed a significant 3-fold increase of colocalization in cells cultured at 10 mmol/liter glucose in comparison to cells cultured at 3 mmol/liter glucose (Fig. 4CGo).


Figure 4
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  		FIG. 4. Effects of glucose on the colocalization between glucokinase and PFK-2/FBPase-2 in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. Cells were seeded on glass cover slips and grown for 24 h in medium with 10 mmol/liter glucose. Thereafter, cells were incubated for the next 24 h in medium with either 3 (A) or 10 (B) mmol/liter glucose. Finally, cells were fixed and double immunostained for glucokinase (green) and PFK-2/FBPase-2 (red). Yellow color indicates colocalization. C, Colocalization was calculated by Image J/ Colocalization Finder (C. Laummonerie, Strasbourg, France). Shown are means ± SEM from three individual experiments. **, P < 0.01 compared with cells cultured at 3 mmol/liter glucose (Student’s t test).

 
Effects of islet PFK-2/FBPase-2 overexpression on glucose utilization in RINm5F-GK cells
Production of 3H2O from D-[5-3H]glucose was measured to estimate the overall rate of glycolysis. Rates of glucose utilization increased in a concentration-dependent manner in RINm5F-GK and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. Glucose utilization rates were significantly higher in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells than in RINm5F-GK cells at 5 and 10 mmol/liter glucose (Fig. 5Go). Thus, islet PFK-2/FBPase-2 overexpression resulted in an enhanced glycolytic flux in insulin-producing cells.


Figure 5
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  		FIG. 5. Effects of islet PFK-2/FBPase-2 overexpression on glucose utilization in RINm5F-GK cells. RINm5F-GK cells (white circles) and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells (black circles) were grown in medium with 10 mmol/liter glucose. After 48 h, cells were incubated for 1 h 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/liter). Glucose utilization was calculated from the production of 3H2O. Shown are means ± SEM from seven individual experiments. *, P < 0.05 compared with RINm5F-GK cells (Student’s t test).

 
ATP/ADP ratio, ATP content, and ADP content of RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells
The intracellular ATP/ADP ratio (Fig. 6AGo) was not different in RINm5F cells cultured at 3 mmol/liter or at 10 mmol/liter glucose due to a comparable ATP and ADP content (Fig. 6Go, B and C). In contrast, glucokinase overexpressing RINm5F-GK cells showed a 14% higher and glucokinase plus islet PFK-2/FBPase-2 co-overexpressing RINm5F-GK-PFK-2/FBPase-2 (I 4) cells a 32% higher ATP/ADP ratio at 10 mmol/liter glucose than at 3 mmol/liter glucose (Fig. 6AGo), mediated by an increase in the ATP content (Fig. 6BGo) and a decrease in the ADP (Fig. 6CGo) content. Although at 3 mmol/liter glucose the ATP/ADP ratio was comparable in control and transfected RINm5F cells, at 10 mmol/liter glucose RINm5F-GK cells and in particular RINm5F-GK-PFK-2/FBPase-2 (I 4) cells showed a significantly higher ATP/ADP ratio than RINm5F cells (Fig. 6AGo). Incubation of control and transfected RINm5F cells at 30 mmol/liter glucose resulted in comparable ATP/ADP ratios as observed for cells cultured at 10 mmol/liter glucose (data not shown).


Figure 6
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  		FIG. 6. ATP/ADP ratio (A), ATP content (B), and ADP content (C) in RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells cultured at 3 and 10 mmol/liter glucose. Cells were grown for 24 h in medium with 10 mmol/liter glucose. Thereafter, cells were incubated for the next 48 h in medium with either 3 (gray bars) or 10 mmol/liter (black bars) glucose. After permeabilization of the cells, ATP and ADP contents were measured by a luminometric assay, and the ATP/ADP ratio was calculated. Shown are means ± SEM from three to five individual experiments. **, P < 0.01 compared with RINm5F cells (ANOVA/Bonferroni’s test).

 
Cell viability of RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells
The viability of RINm5F cells was not affected by incubation for 48 h at 3 mmol/liter glucose (105 ± 12%) or at 30 mmol/liter glucose (94 ± 9%) in comparison to 10 mmol/liter glucose (100 ± 12%) in the culture medium. RINm5F-GK cells and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells also showed no difference in the cell viability after incubation at 3 mmol/liter glucose (99 ± 9 and 97 ± 9%) or 30 mmol/liter glucose (98 ± 6 and 97 ± 10%) compared with 10 mmol/liter (data not shown). Thus, neither glucokinase nor glucokinase plus islet PFK-2/FBPase-2 overexpression provoked glucose toxicity in RINm5F cells. Glucokinase enzyme activity was differentially regulated by high glucose concentrations in the culture medium in RINm5F-GK and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. RINm5F-GK-PFK-2/FBPase-2 (I 4) cells cultured at 30 mmol/liter glucose showed 15% higher glucokinase enzyme activities in comparison to cells incubated at 10 mmol/liter glucose. In contrast, glucokinase enzyme activity in RINm5F-GK cells was reduced by 10% in cells cultured at 30 mmol/liter glucose in comparison to cells incubated at 10 mmol/liter glucose (data not shown).

Effects of glucokinase overexpression and glucokinase plus islet PFK-2/FBPase-2 co-overexpression on pyruvate and lactate production in RINm5F cells
For further characterization of glucose metabolism, RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells were cultured for 48 h at 10 or 30 mmol/liter glucose (Fig. 7Go). Both lactate and pyruvate production increased, dependent on the glucose concentration in all cell types. Although pyruvate production was comparable in RINm5F and RINm5F-GK cells cultured at 10 mmol/liter glucose (Fig. 7AGo), the lactate production of RINm5F cells was slightly higher, with significantly higher lactate values at glucose concentrations of 0.5 and 2 mmol/liter in comparison to RINm5F-GK cells (Fig. 7BGo). Pyruvate and lactate production was significantly lower in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells than in RINm5F-GK cells cultured at 10 mmol/liter glucose (Fig. 7Go, A and B). Pyruvate production was decreased in RINm5F and RINm5F-GK cells but not in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells at 30 mmol/liter glucose in comparison to 10 mmol/liter glucose. Thus, at high glucose concentration, the pyruvate production in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells was lower, but not significantly different from RINm5F-GK cells (Fig. 7CGo). Lactate production increased in all cells after incubation at 30 mmol/liter glucose. RINm5F cells showed in comparison with RINm5F-GK cells significantly lower lactate values at glucose concentrations of 5 and 10 mmol/liter (Fig. 7DGo). Notably, lactate production in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells was significantly lower at all glucose concentrations when compared with RINm5F-GK cells (Fig. 7DGo).


Figure 7
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  		FIG. 7. Pyruvate and lactate production in RINm5F, RINm5F-GK, and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells. Measurements were performed in RINm5F cells (black squares/dashed line), RINm5F-GK cells (white circles/closed line), and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells (black circles/closed line). Cells were either grown for 72 h in medium with 10 mmol/liter glucose (A, B) or grown for 24 h in medium with 10 mmol/liter glucose and for the next 48 h in medium with 30 mmol/liter glucose (C, D). Thereafter, cells were incubated for 1 h at 37 C and 300 rpm in Krebs-Ringer buffer in the presence of various glucose concentrations (0.5, 1, 2, 5, and 10 mmol/liter). After centrifugation lactate and pyruvate contents were measured spectrophotometrically in the supernatant. Shown are means ± SEM from four to seven individual experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with RINm5F-GK cells (ANOVA/Bonferroni’s test).

 
Insulin secretion in RINm5F-GK cells overexpressing islet PFK-2/FBPase-2
Although RINm5F cells showed a basal insulin secretion rate of 18.5% in relation to insulin content and no demonstrable insulin secretory response to glucose (data not shown), glucokinase overexpressing RINm5F-GK cells as well as glucokinase plus islet PFK-2/FBPase-2 co-overexpressing RINm5F-GK-PFK-2/FBPase-2 (I 4) cells were responsive to glucose (Fig. 8Go). RINm5F-GK and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells exhibited basal insulin secretion rates of 10.9 and 11.1%, respectively, and a comparable insulin secretion at 0 and 1 mmol/liter glucose (Fig. 8Go). Notably, however, RINm5F-GK-PFK-2/FBPase-2 (I 4) cells showed a higher rate of insulin secretion than RINm5F-GK cells in response to 3, 5, 10, and 25 mmol/liter glucose (Fig. 8Go). Insulin secretion in response to KCl (25 mmol/liter), a nonnutritional stimulus, was not different in both cell types, and insulin content was not affected by overexpression of islet PFK-2/FBPase-2 in RINm5F-GK cells (data not shown).


Figure 8
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  		FIG. 8. Effects of islet PFK-2/FBPase-2 overexpression on insulin secretion from RINm5F-GK cells. RINm5F-GK cells (white bars) and RINm5F-GK-PFK-2/FBPase-2 (I 4) cells (black bars) were incubated for 1 h at 37 C without or in the presence of various glucose concentrations (1, 3, 5, 10, and 25 mmol/liter). Thereafter, insulin was measured in the supernatant. Shown are means ± SEM from four to six individual experiments. *, P < 0.05 compared with RINm5F-GK cells (ANOVA/Bonferroni’s test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Regulation of the glucose sensor enzyme glucokinase in pancreatic ß-cells is complex and only partially understood. Posttranslational mechanisms are important for the adaptation of the glucokinase enzyme activity to the physiological requirements of the ß-cell (6, 7, 11). Although glucose is the main stimulus in the process of glucokinase regulation (2, 5) other modulators are crucial to confer a precise adaptation of glucokinase enzyme activity to the needs of metabolic stimulus-secretion coupling. Intracellular structures such as insulin secretory granules and soluble proteins have been considered as potential regulators of glucokinase activity (10, 11, 12, 13, 14, 20, 31, 32). The bifunctional enzyme PFK-2/FBPase-2 is a cytoplasmic binding partner of glucokinase, which posttranslationally activates the enzyme in ß-cells (18, 20). Overexpression of PFK-2/FBPase-2 resulted in a significant increase in glucokinase enzyme activity in insulin-producing RINm5F-GK and INS1 cells (18). Our present results indicate that, besides the physiological up-regulation of glucokinase through endogenous PFK-2/FBPase-2 in ß-cells, a further activation can be achieved by an additional expression of PFK-2/FBPase-2. Thus, the glucokinase enzyme activity level correlates with the PFK-2/FBPase-2 protein expression level. In islet PFK-2/FBPase-2 overexpressing insulin-producing RINm5F-GK cells, down-regulation of PFK-2/FBPase-2 by siRNA resulted in a concomitant decrease of glucokinase enzyme activity.

Because of the metabolic key role of glucokinase, changes in the enzyme activity status will directly affect the glycolytic flux and the initiation of insulin-secretion in pancreatic ß-cells. This pivotal function is in particular illustrated in patients carrying activating or inactivating glucokinase gene mutations (7, 8, 33). As activation of glucokinase is a promising concept for treatment of type 2 diabetes, this enzyme proved to be an interesting target for antidiabetic drugs (15, 16, 17). The recently developed small chemical compounds activate glucokinase by binding to an allosteric site of the enzyme protein. The glucokinase activators RO281675, RO0274375, and LY2121260 are especially interesting molecules, because they are able to increase the Vmax value of glucokinase along with a decrease in the glucose S0.5 (15, 17, 34). A sole decrease of the S0.5 for glucose would result in an undesirable left-shift in the characteristic sigmoidal response curve of the enzyme. Thus, an increase of the Vmax value of glucokinase, which improves the sensor function within the millimolar glucose concentration range, is a major goal of glucokinase activation (34). Interestingly, glucokinase activation through PFK-2/FBPase-2 mirrors the physiological regulation with an increase in the Vmax value, whereas the S0.5 for glucose remains unchanged (18). The activation of glucokinase enzyme activity by PFK-2/FBPase-2 improved metabolic stimulus-secretion coupling in insulin-producing cells (18). Importantly, the glucokinase activators RO281675, RO0274375, and LY2121260 induced a stimulation of glucose-induced insulin secretion in rat pancreatic islets (15, 17, 34).

It is well known that a reasonable overexpression of glucokinase protein in ß-cells increases glucokinase enzyme activity and concomitantly glucose metabolism (21, 35). However, it is not clear whether high amounts of glucokinase protein will have the same effect upon glucose metabolism in ß-cells as activation of constitutive physiological glucokinase protein levels. Activated glucokinase is present in the closed conformation and prevents the conformational transition to the super-open form as shown by crystallographic analyses (9). This is an important aspect based on the fact that impaired insulin secretion and a loss of cell viability have been described in insulin-producing cells after an excessive overexpression of glucokinase (36). Moreover, recently an increase in oxidative stress has been reported to aggravate glucose toxicity in insulin-secreting cells even after moderate overexpression of glucokinase (37).

In the present study, glucose metabolism has been analyzed in RINm5F-GK cells overexpressing the endogenous glucokinase activator PFK-2/FBPase-2 and compared with RINm5F-GK and RINm5F cells. These experiments have been performed in insulin-producing cells with the islet PFK-2/FBPase-2 isoform, which, in contrast to the liver isoform, is not regulated by phosphorylation and dephosphorylation (20, 38). Changes in the F-2,6-P2 level obtained by overexpression of another PFK-2/FBPase-2 in RINm5F-GK cells, namely the liver or liver mutant isoform, revealed differences to the islet PFK-2/FBPase-2 isoform. Nevertheless, however, this overexpression in insulin-producing cells cannot mirror the metabolic regulation in hepatocytes. Clearly, control of the cellular F-2,6-P2 concentration and glucokinase regulation by PFK-2/FBPase-2 in ß-cells differ from the regulatory principles in liver (39, 40, 41, 42). In ß-cells, F-2,6-P2 is of a subordinate physiological relevance, and the enzyme activity is dominated by the PFK-2 domain (43, 44). Our present results show that overexpression of the islet PFK-2/FBPase-2 isoform did not affect the regulation of the cellular F-2,6-P2 concentrations and thus had no direct effect on glucose metabolism in insulin-producing cells.

The activation of glucokinase by PFK-2/FBPase-2 proved to be glucose dependent. Overexpression of islet PFK-2/FBPase-2 in RINm5F-GK cells resulted in a greater stimulatory effect of glucose on glucokinase enzyme activity when the glucose concentration was increased from 2 to 10 mmol/liter (18). Importantly, our present results demonstrate also a glucose-dependent increase in the colocalization between glucokinase and PFK-2/FBPase-2 in these cells. Thus, there is evidence that the binding site of glucokinase is mainly accessible for an interaction with PFK-2/FBPase-2 in the closed conformation of the enzyme when the substrate glucose is bound (9). This may explain why activation of glucokinase by PFK-2/FBPase-2 affects only the Vmax of the enzyme, but not the S0.5 for glucose, and has to be elucidated in further experiments.

Overall glucose metabolism is up-regulated in RINm5F-GK cells overexpressing the glucokinase activator PFK-2/FBPase-2. Thus, the increase in glucokinase enzyme activity not only enhances glucose phosphorylation but also improves significantly the coupling between glycolysis and oxidative flux. Moreover, this improved metabolic coupling could also be demonstrated by a lesser accumulation of pyruvate and conversion to lactate in RINm5F-GK cells overexpressing PFK-2/FBPase-2 both in normal and high glucose culture. It should be noted that RINm5F cells exhibit high lactate dehydrogenase activities, thereby provoking anaerobic conversion of pyruvate into lactate (25). The improved metabolism in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells is also demonstrated by an increase in the ATP/ADP ratio at 10 mmol/liter glucose compared with RINm5F-GK and RINm5F cells. In control RINm5F cells, glucose metabolism is dominated by considerable levels of high-affinity hexokinases (21). With glucokinase overexpression in RINm5F-GK cells, the ß-cell characteristic responsiveness to millimolar glucose concentration could be established. RINm5F-GK cells showed both significantly lower basal insulin release than RINm5F cells and a threshold for glucose-induced metabolism, which is somewhat lower than in ß-cells probably due to the high levels of low-Km hexokinases. Nevertheless, RINm5F-GK cells showed an increase in the ATP/ADP ratio as well as in glucose-stimulated insulin secretion in dependence on the millimolar glucose concentration. Importantly, additional overexpression of PFK-2/FBPase-2 in RINm5F-GK-PFK-2/FBPase-2 (I 4) cells caused a doubling of glucokinase activities with concomitant increases of the metabolic flux rates without any effect upon cell viability. This resulted in a higher ATP/ADP ratio and in a significant increase in glucose-stimulated insulin secretion compared with RINm5F-GK cells. This is in accordance with a report that the glucokinase activator RO0281675 augmented cellular respiration in parallel with the enhancement of glucose-stimulated insulin secretion (34).

In contrast to the liver, the glucose sensor function in ß-cells is dependent upon relatively low glucokinase activities whose controlled activation through PFK-2/FBPase-2 or small chemical compounds seems to have a more integrative effect on glucose metabolism than excessive overexpression of the enzyme. Probably, the closed conformation of glucokinase may play a pivotal role in the macromolecular channeling of glycolytic intermediates. Metabolic compartmentation is a generally accepted biological phenomenon (45, 46, 47), but due to its great complexity its details are not well elucidated. Based on conformational data (9), it seems to be plausible that stabilized glucokinase is favorable for dynamic complex formation in the channeling process.

The present study supports the assumption that activation of glucokinase through interaction with the bifunctional enzyme PFK-2/FBPase-2 is an important element of posttranslational glucokinase regulation in pancreatic ß-cells (8, 48, 49, 50). Furthermore, glucokinase activation is an attractive therapeutic strategy, and promising activating small chemical compounds have been described recently (15, 17, 34). The beneficial effect on key metabolic parameters in pancreatic ß-cells by PFK-2/FBPase-2-mediated glucokinase activation will contribute to a better understanding of ß-cell glucokinase regulation and promote further studies on the metabolic mechanisms underlying coupling between glucokinase activation and glucose-induced insulin secretion.


   Acknowledgments
 
The skillful technical assistance of M. Boeger and B. Lueken is gratefully acknowledged.


   Footnotes
 
This work has been supported by the German Diabetes Association and the Dr. Buding Foundation (to S.B.). L.M. was a recipient of a grant from the Ministry of Science and Culture of Lower Saxony, on leave from the CENEXA Center of Experimental and Applied Endocrinology, National University of La Plata School of Medicine, La Plata, Argentina.

Disclosure statement: The authors have nothing to declare.

First Published Online September 15, 2006

Abbreviations: F-2,6-P2, Fructose-2,6-bisphosphate; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; PFK-2/FBPase-2, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; siRNA, small interfering RNA; TxRed, Texas Red.

Received May 24, 2006.

Accepted for publication September 6, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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