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J. Biol. Chem., Vol. 276, Issue 4, 2325-2328, January 26, 2001

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ACCELERATED PUBLICATION
A Novel Glucokinase Regulator in Pancreatic  Cells

PRECURSOR OF PROPIONYL-CoA CARBOXYLASE  SUBUNIT INTERACTS WITH GLUCOKINASE AND AUGMENTS ITS ACTIVITY^*

Akihiko Shiraishi^§, Yuichiro Yamada, Yoshiyuki Tsuura, Shimpei Fijimoto, Katsushi Tsukiyama, Eri Mukai, Yukiyasu Toyoda^¶, Ichitomo Miwa^¶, and Yutaka Seino

From the  Department of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto University, Sakyoku, Kyoto 606-8507 and the ^¶ Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University, Tempaku-ku, Nagoya 468-8503, Japan

Received for publication, August 7, 2000, and in revised form, November 11, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A glucokinase regulatory protein has been reported to exist in the liver, which suppresses enzyme activity in a complex with fructose 6-phosphate, whereas no corresponding protein has been found in pancreatic  cells. To search for such a protein in pancreatic  cells, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat islet by the yeast two hybrid system. We detected a cDNA encoding the precursor of propionyl-CoA carboxylase  subunit (pPCCase), and glutathione S-transferase pull-down assay illustrated that pPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts. Functional analysis indicated that pPCCase decreased the K_m value of recombinant islet glucokinase for glucose by 18% and increased V_max value by 23%. We concluded that pPCCase might be a novel activator of glucokinase in pancreatic  cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glucose phosphorylation is known to be the rate-limiting step in glycolysis of pancreatic  cells (1). This step is catalyzed by hexokinase through a low K_m pathway and by glucokinase through a high K_m pathway (2, 3). Glucokinase predominantly regulates intracellular glucose metabolism when ambient glucose concentration is increased (4), and the reduced activity of this enzyme has been reported to decrease glucose-induced insulin release (5). Glucokinase is also expressed in the liver, which is another regulatory center of glucose homeostasis. Van Schaftingen et al. (6) have reported that the rat liver contains a regulatory protein that binds to glucokinase, and the activity of glucokinase coupled to this protein is thought to be mediated by competitive binding of fructose 6-phosphate and fructose 1-phosphate in the liver (6, 7). Thus, this feedback regulation may affect the balance of glycolysis and gluconeogenesis. There have also been attempts to find a corresponding protein in pancreatic islets (8). Fructose 1-phosphate converted from fructose was reported to enhance glucokinase activity in vitro (9), and a greater increase in glucokinase activity has been reported in extracts from islets overexpressing glucokinase without an increase of glucose utilization (10). These reports suggested the existence of a factor capable of regulating glucokinase activity or its affinity for glucose under physiological intracellular conditions, although as yet, no direct evidence for such a molecule has been reported. To clarify whether a glucokinase regulatory protein is present in pancreatic islets, we screened for a cDNA library of the HIT-T15 cell line with the cDNA of glucokinase from rat pancreatic  cells using the yeast two hybrid system and found a novel binding regulator for glucokinase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning-- Rat pancreatic islet glucokinase full-length cDNA was amplified by polymerase chain reaction (PCR)¹ using pancreatic  cell glucokinase-specific oligonucleotides (11). This cDNA was subcloned into the following two types of plasmids: pBTM116, the "bait" for yeast two hybrid screen (pBTM116-GK), and pGEX4T-1 (Amersham Pharmacia Biotech) to produce glutathione S-transferase (GST)-fused glucokinase (GST-GK). Full-length cDNA of precursor of propionyl-CoA carboxylase  subunit (pPCCase) in pancreatic islet was also amplified from rat pancreatic islets cDNA by PCR using the oligonucleotides for rat liver pPCCase (12).

Recombinant Protein-- Rat pancreatic islet glucokinase and pPCCase were subcloned into pGEX4T-1 and transformed into Escherichia coli strain M15. Following induction of protein expression with isopropyl thio--D-galactosidase, the GST fusion proteins were purified according to the manufacturer's instructions (Amersham Pharmacia Biotech) (13). The produced protein was dissolved in 50 mM Tris-HCl, pH 8.0. Recombinant glucokinase without GST tag (rGK) was made by thrombin cleavage according to the manufacturer's instruction.

Screening for the cDNA Encoding a Protein with Affinity for Rat Islet Glucokinase by the Yeast Two Hybrid System-- Yeast two hybrid screening was performed as described previously (14). The bait plasmid was pBTM116-GK, in which rat pancreatic  cell glucokinase was fused to the LexA DNA-binding domain. The yeast strain L40 (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-LacZ) was transformed with pBTM116-GK using the lithium acetate method (15). Strain L40 carrying pBTM116-GK was transformed with a HIT-T15 (hamster pancreatic  cell line (16)) cell cDNA library. Approximately 1.0 × 10⁶ transformants were screened for growth on Yc plate medium (2% glucose, 0.5% ammonium sulfate, 1% succinic acid, and 0.12% yeast nitrogen base) containing 0.5 mM 3-amino-1,2,4-triazole but lacking tryptophan, histidine, uracil, and leucine. His⁺ colonies were then placed on paper filters and stained with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside for -galactosidase activity as described (15). Two His⁺ and LacZ⁺ clones were obtained with this screening. The plasmids were transformed to E. coli and isolated.

Preparation of Rat Pancreatic Islet and Liver Extract-- Pancreatic islets were isolated from male Wistar rats by collagenase digestion as described previously (17) and were then sonicated in RIPA (150 mM NaCl, 1.0% Nonidet P-40, 50 mM Tris-HCl, pH 8.0). The homogenate was centrifuged at 10,000 × g for 15 min at 4 °C, and the supernatant was collected as the islet extract. Rat liver tissue was also homogenized in RIPA, and the supernatant was collected after centrifugation at 10,000 × g for 15 min at 4 °C as liver extract.

Preparation of Islet Cellular Fractions-- Sucrose gradient fractionation was performed for collected islets by the previously described method (18). Islets in the 0.25 M sucrose were homogenized in a Potter-Elvehjem homogenizer with 6 strokes of a close-fitting Teflon pestle. The mixture was centrifuged for 10 min at 600 × g. The pellet was further purified by the method of Blobel-Potter to obtain the purified nuclear fraction (19). The supernatant was centrifuged for 10 min at 8,000 × g. The pellet was collected as the mitochondrial fraction, and the supernatant was centrifuged for 60 min at 105,000 × g. The resultant pellet was the microsomal fraction, and the supernatant was the cytosolic fraction. Each subcellular fraction was analyzed by SDS-PAGE and Western blotting with anti-pPCCase serum as described previously (13). The anti-pPCCase serum was made from a rabbit immuned with GST-pPCCase.

In Vitro Protein Interaction Assays-- GST pull-down assay was performed as described previously (13). Each assay contained GST fusion protein immobilized on glutathione-Sepharose beads (Amersham Pharmacia Biotech). Briefly, samples containing glucokinase (5 µg of rGK, rat pancreatic islet extract, or liver extract) were incubated with 3 µg of GST (molecular mass, 27 kDa) or 9 of µg GST-pPCCase (molecular mass, 89 kDa) or 8 µg of GST-GK (molecular mass, 79 kDa) in binding buffer (20 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 20% glycerol, 300 mM KCl, 0.1% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM MgCl₂, and 5 mM 2-mercaptoethanol) for 1 h at 4 °C. After incubation, the beads were washed five times with binding buffer (10-fold volume of beads) and resuspended in elution buffer (binding buffer containing 10 mM reduced glutathione). The bound proteins were resolved by 10% SDS-PAGE and visualized by Western blotting with rabbit anti-glucokinase antibody or anti-pPCCase serum as described previously (13, 20).

Assay for Glucokinase Activity-- Glucokinase enzyme activity was measured by a fluorometric assay according to the method reported previously (21). Glucokinase activity was measured as the fluorescence of NADPH at excitation and emission wavelengths of 340 and 450 nm, respectively. The reaction was performed in a solution consisting of 50 mM HEPES-NaOH, pH 7.4, 100 mM KCl, 8 mM MgCl₂, 0.5 mM NADP, 1 mM dithiothreitol, 1 unit/ml of glucose 6-phosphate dehydrogenase, ATP at various concentrations, and 0.2 µg/ml of GST-GK supplemented with various concentrations of glucose at 37 °C for half an hour and was stopped by addition of stop solution (300 mM NaH₂PO₄, 0.46 mM sodium dodecylsulfate, pH 8.0). Reagent blanks were incubated in the absence of ATP and were subtracted from the total fluorescence of the corresponding complete reaction mixtures. The K_m, V_max, and Hill coefficient were determined by the nonlinear least squares routine to fit the Hill equation using Sigma Plot (1, 9).

Statistical Analysis-- All data are expressed as means ± S.E., with n values being the number of experiments performed. Statistical significance was determined by paired Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The yeast two hybrid system was used to identify cDNAs in the HIT-T15 cell cDNA library encoding the regulatory protein using the bait plasmid pBTM116-GK. The resultant cDNA insert was isolated by nutritional selection and -galactosidase activity and was shown to be 68% of the rat liver pPCCase gene by sequence analysis. The full-length cDNA of pPCCase was obtained from rat pancreatic islet cDNA by a PCR-based method. The specificity of the interaction between pancreatic islet glucokinase and pancreatic islet pPCCase was tested in vitro. The GST-fused pPCCase (GST-pPCCase) could bind to rGK and glucokinase in cellular extracts of rat pancreatic islets and liver (Fig. 1). Furthermore, the GST-fused glucokinase (GST-GK) could bind to pPCCase in cellular extracts of rat pancreatic islets (Fig. 1D).

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Interaction of pPCCase with GK. A, interaction of GST-pPCCase with recombinant GK. B, interaction of GST-pPCCase with cellular extract of pancreatic islets. C, interaction of GST-pPCCase with cellular extract of liver. D, interaction of GST-GK with cellular extract of pancreatic islets. GST-pPCCase interacted with GK, and GST-GK interacted with pPCCase in vitro. GST or GST-pPCCase, immobilized on glutathione beads, was incubated with recombinant GK, rat pancreatic islet, or liver cellular extract. GST-GK immobilized on glutathione beads was also incubated with islet extract. Bound proteins were detected with anti-GK antibodies or anti-pPCCase serum.

Western blotting was performed to confirm the subcellular localization of pPCCase in islets. pPCCase was shown to be present in the mitochondrial fraction and cytosolic fraction, but it was not detected in the microsomal or nuclear fractions (Fig. 2).

View larger version (57K): [in this window] [in a new window]   Fig. 2.   pPCCase in various islet cell fractions. Western blotting with anti-pPCCase serum is shown. pPCCase was visualized in the cytosolic fraction and mitochondrial fraction. Each lane contained 1 µg of protein.

Next, the effects of GST-pPCCase on glucokinase activity were investigated. When 25 µg/ml GST-pPCCase was added, GST-GK activity was significantly higher than that in controls within the range of 3 to 50 mM glucose (Fig. 3A), whereas the GST tag alone did not show any effect on GST-GK activity (data not shown). GST-pPCCase had no activity of hexokinase or glucokinase. The glucokinase inhibitor mannoheptulose at 20 mM suppressed the activity of GST-GK by 67 or 60% in the presence or absence of pPCCase, respectively, when the activity was assayed in the presence of 10 mM glucose and 5 mM ATP. The V_max value of GST-GK for glucose was increased by about 23%, and the K_m value was reduced by 18% when GST-pPCCase was added (Table I). The Hill number was 1.8 in the presence of GST-pPCCase, which was not significantly different from the value of 1.7 in controls (Table I). Furthermore, the K_m value of GST-GK for ATP was not affected by addition of GST-pPCCase (see Fig. 3B and Table I). The activity of GST-GK was increased in a GST-pPCCase concentration-dependent manner (Fig. 3C). In the previous reports, palmitoyl-CoA has been shown to suppress glucokinase activity at higher concentrations beyond physiological ones (22-24). We tested the effects of pPCCase on glucokinase activity in the presence or absence of palmitoyl-CoA and propionyl-CoA. The activating effect of GST-pPCCase was not influenced by the presence of 3 µM palmitoyl-CoA and 3 µM propionyl-CoA (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effects of pPCCase on GK activity. A, reaction velocity of GST-GK (closed circles) or GST-GK + 25 mg/ml GST-pPCCase (open circles) with glucose concentrations from 0.05 to 50 mM at 5 mM ATP (n = 5). B, reaction velocity of GST-GK (closed circles) or GST-GK + GST-pPCCase (open circles) with ATP concentrations from 0.01 to 10 mM at 5 mM glucose (n = 4). C, reaction velocity of GK with various concentrations of pPCCase (up to 30 µg/ml) at 10 mM glucose and 5 mM ATP (n = 5). Values shown are means ± S.E. from four or five independent experiments. * indicates p < 0.01 (or 0.05) versus each control value.

View this table: [in this window] [in a new window]   Table I Kinetic parameters of GST-GK activity in the presence or absence of GST-pPCCase h, Hill coefficient number; NS, not significant. The data are expressed as means ± S.E. Km, Vmax, and h were calculated from six independent experiments, and Km(ATP) was calculated from four independent experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we found that pPCCase, which is present in the cytosolic compartment (25-27), unexpectedly interacted with pancreatic islet glucokinase and activated its enzyme activity with increased V_max and decreased K_m values for glucose.

Liver and pancreatic  cells play crucial roles in glucose homeostasis, but they have distinctive functions (28). In the liver, in response to changes in plasma glucose concentration, hepatic glucose uptake and glucose output are alternatively increased to maintain the plasma glucose concentration. Accordingly, an abrupt alteration of ambient glucose concentration might require a negative feedback loop for glucokinase activity by binding of a regulatory protein coupled to fructose 6-phospate (6, 29). On the other hand, pancreatic  cells secrete insulin in response to increasing glucose concentration (4). A glucose-induced insulin secretion is subject to intracellular glucose metabolism, and in  cells glucose metabolism unidirectionally flows toward the direction of ATP production with the metabolic characteristic of development of a glycerol phosphate shuttle (30), lack of fructose-bisphosphatase (31), and a low activity of lactate dehydrogenase (32). However, a positive regulator of glucokinase, which has the lowest activity among all the glycolytic enzymes, had not been found in pancreatic  cells. In the present study, we demonstrated that pPCCase increased V_max of glucokinase for glucose and reduced K_m. Such regulation may enable maximal enhancement of glucose metabolism in the range of physiological ambient glucose concentrations.

pPCCase is a precursor of the  subunit in propionyl-CoA carboxylase (PCCase), a biotin-dependent enzyme that is present in the matrix of mitochondria (25-27). pPCCase protein (61.5 kDa) is known to be synthesized on cytoplasmic polyribosomes and transferred to the mitochondria via a specific transporter. After arrival at the mitochondrial matrix, pPCCase is processed to mature  subunit of propionyl-CoA carboxylase protein (54 kDa) by a Zn²⁺-dependent proteolytic component in the matrix (27). Thus, pPCCase could exist in the cytoplasmic compartment. The importance of the regulation of glucokinase activity by pPCCase remains to be determined. Pancreatic  cells are continuously exposed to not only glucose but also fatty acids, which are known to affect glucose-induced insulin secretion (33, 34). Palmitate and oleate are strong potentiators of glucose-induced insulin release (35, 36), and Liu et al. (33) reported that glucose utilization examined using [²H]glucose was augmented accompanied by a decrease in the level of glucose 6-phosphate in islets cultured with oleate. These findings seemed to be consistent with the hypothesis that a precursor of PCCase  subunit, which exists in the cytosolic compartment and is known to become as functional as PCCase in fatty acid metabolism in the mitochondrial matrix (36, 37), enhances the glycolytic pathway by up-regulating glucokinase activity. This hypothesis might be applicable to the glucose metabolism in the liver. In addition, pPCCase might contribute to the localization of glucokinase by transporting the coupled enzyme in the liver and pancreatic  cells.

In conclusion, pPCCase has been shown to be a novel activator of glucokinase. Unlike the binding regulatory protein in the liver (7, 6), pPCCase alters the K_m and V_max values of glucokinase for glucose, resulting in enhanced enzyme activity in islets. Because glucokinase is a rate-limiting enzyme (28), pPCCase may be directly involved in the regulation of glucose metabolism and glucose-induced insulin secretion in pancreatic islets.

	ACKNOWLEDGEMENTS

We thank Susumu Seino of Chiba University Graduate School of Medicine for arranging the yeast two hybrid screening system.

	FOOTNOTES

^* This study was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, Grant-in-Aid for Creative Basic Research 10NP0201 from the Ministry of Education, Science, Sports and Culture of Japan, and grants from the Research for the Future Program of the Japan Society for the Promotion of Science (JSPS-RFTF97I00201).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Metabolism and Clinical Nutrition, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawahara-cho, Sakyoku, Kyoto 606-8507, Japan. Tel.: 81-75-751-3560; Fax: 81-75-751-4244; E-mail: siraisi@metab.kuhp. kyoto-u.ac.jp.

Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.C000530200

	ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; GST, glutathione S-transferase; GK, glucokinase; pPCCase, precursor of propionyl-CoA carboxylase  subunit; r, recombinant; PCCase, propionyl-CoA carboxylase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 276/4/2325    most recent C000530200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Shiraishi, A. Articles by Seino, Y. Search for Related Content PubMed PubMed Citation Articles by Shiraishi, A. Articles by Seino, Y. Social Bookmarking                      What's this?