A Novel Glucokinase Activator Modulates Pancreatic Islet and Hepatocyte Function
http://www.100md.com
《内分泌学杂志》
Lilly Research Laboratories (A.M.E., D.G.B., M.B.B., U.G., M.R., G.S.G., S.S., A.W., A.Z., J.G.), 22419 Hamburg, Germany
Lilly Research Laboratories (S.L.B., J.D.D., H.G., Y.W.), Indianapolis, Indiana 46285
Lilly Development Centre (A.D.), 1348 Mont-Saint-Guibert, Belgium
Abstract
The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic -cells and increased glucose use in rat hepatocytes. In addition, incubation of -cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.
Introduction
GLUCOKINASE (GK), A MEMBER of the hexokinase family, catalyzes the first step in glycolysis, phosphorylation of glucose to glucose 6-phosphate. It is a unique hexokinase in that it has a low affinity for glucose, demonstrates non-Michaelis-Menten kinetics (Hill coefficient is around 1.5) and displays no inhibition by the product of the reaction, glucose 6-phosphate (1). These particular features make GK an ideal sensor of physiological changes in blood glucose levels, translating them into changes in the metabolic status of the cell.
GK has a limited tissue distribution and is expressed predominantly in metabolically active tissues: brain, gut, liver, and pancreas. The roles of GK in pancreas and liver are well understood. In pancreatic -cells, GK is the rate-limiting enzyme in glucose metabolism and determines the rate of glucose-induced insulin secretion (1, 2). In liver, GK activity determines the rate of glucose use and glycogen synthesis (3). These roles make GK the critical player in the regulation of blood glucose levels. Activation of the enzyme at postprandial glucose levels produces a rise in insulin secretion from the pancreas as well as stimulation of glucose uptake and glycogen synthesis in liver, the combination of which leads to an increase in glucose disposal and the return of blood sugar to basal levels.
The significance of GK in the control of blood glucose is underscored by data from animal models, in which GK activity has been modulated, and in humans possessing GK mutations. GK-deficient mice die early after birth due to severe diabetes (4, 5). Pancreatic -cell or liver-specific GK knockout mice also display hyperglycemia, the severity of the phenotype being more pronounced in -cell knockouts (5). On the other hand, overexpression of GK leads to lower basal blood glucose levels and resistance to the development of high-fat-induced diabetes (6, 7). In humans, more than 150 naturally occurring mutations in GK have been reported to date (8). It has been shown that mutations in a single GK gene allele that impair enzyme function cause type 2 maturity onset diabetes of young (MODY2) (9), whereas inactivating mutations in both GK alleles cause permanent neonatal diabetes (10). In addition, GK mutations that increase enzyme activity cause hyperinsulinemic hypoglycemia (11).
Recently small molecules that activate GK via binding to an allosteric site on the enzyme have been discovered (12, 13, 14). Through their ability to enhance the activity of GK, such so-called GK activators hold promise as a novel and effective treatment for type 2 diabetes. Herein we describe the effects of a novel GK activator in pancreatic -cells and hepatocytes.
Materials and Methods
Glucokinase enzymatic assay
An enzymatic GK assay using purified recombinant human islet GK was used to evaluate the effects of potential small molecule activators. In the assay, GK catalyzes glucose phosphorylation in the presence of ATP. The product of this reaction, glucose-6-phosphate, is then oxidized by an excess of glucose-6-phosphate dehydrogenase to produce gluconate-6-phosphate with concomitant reduction of nicotinamide adenine dinucleotide (15). Production of reduced nicotinamide adenine dinucleotide results in an increase in fluorescence, which was used to monitor GK activity. Human islet GK was expressed in Escherichia coli as a (His)6-tagged fusion protein and was purified via metal chelate affinity chromatography (16). The assay was performed in 96-well plates in a final incubation volume of 100 μl. The incubation mixture consisted of (in millimoles) 50 HEPES, 100 KCl, 5 MgCl2, 5 dithiothreitol (DTT), 10 Mg-ATP, 1 nicotinamide adenine dinucleotide (pH 7.4), 4 U/ml glucose-6-phosphate dehydrogenase, 100 ng/ml GK, and glucose at different concentrations.
GK crystal structure
Human GK was expressed as a glutathione-S-transferase fusion protein in E. coli and purified by affinity chromatography, followed by Factor Xa digestion to remove the glutathione-S-transferase and final gel filtration chromatography. Compound LY2121260 was added to GK protein solution (12 mg/ml) containing 20 mM HEPES (pH 7.6), 50 mM NaCl, 10 mM DTT, 50 mM glucose, and 1–2 mM DTT before crystallization. Crystals were grown at 4 C by the hanging drop method under the reservoir condition 0.1 M sodium citrate (pH 5.4), 20% polyethylene glycol 3350, and 0.2 M NH4I. Crystals belong to the space group P4(1)2(1)2 with unit cell parameters a = 70.43 , b = 70.43 , c = 188.75 . The diffraction data (resolution of 2.0 , Rmerge = 0.124, completeness of 96.5%) were collected using a Rigaku RU300 generator and an R-AXIS IV detector (Rigaku/MSC, Inc., The Woodlands, TX). The diffraction data were processed using HKL2000 (HKL Research, Inc., Charlottesville, VA). The crystal structure was determined by the method of molecular replacement (amore from the CCP4 Suite) (17) using coordinates from a previously determined GK complex (Wang, Y., unpublished results), which was determined by molecular replacement with the program CNX2000 (Accelrys, Inc., San Diego, CA) using the human brain hexokinase I structure 1HKB (18) as a search model. The program suite QUANTA 2000 (Accelrys) was used for model building and refinement was carried out with the program CNX2000.
Preparation and culture of pancreatic -cells
Rat pancreatic islets were isolated from male Wistar rats (250–300 g, Charles River Laboratories, Sulzfeld, Germany) as previously described (19). Islets were incubated in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.
INS-1E cells were cultured in RPMI 1640 supplemented with 5% (vol/vol) heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin as described (20).
Insulin secretion assay
Insulin secretion in rat pancreatic islets was performed as described previously (19). For measurements of insulin secretion in INS-1E cells, cells were plated in 96-well plate at density of 20,000 cells/well and cultured for 2 d. Cells were preincubated at 37 C for 30 min in Earle’s balanced salt solution containing 0.1% BSA and no glucose. After preincubation cells were cultured for 1 h in the same buffer containing 5 mM glucose and different concentrations of the tested molecule. Insulin concentration in the collected supernatant was measured with ELISA using rat insulin antibody (Linco Research, St. Charles, MO).
Isolation and culture of hepatocytes
Hepatocytes were isolated from overnight-fasted male Wistar rats by a two-step in situ liver perfusion. Rats were anesthetized by 60 mg/kg pentobarbital. The hepatic portal vein was cannulated, and in a first perfusion step, the liver was flushed with 30 ml/min liver perfusion medium (Invitrogen) for 6 min. In a second step, the liver was digested by perfusion with liver perfusion medium supplemented with 5 mM NaCl, 5 mM CaCl2, and 23.3 mg/liter Liberase Blendzyme 3 (Roche Diagnostics, Mannheim, Germany) at a rate of 15 ml/min for approximately 9 min. The liver was then dissected and transferred to a dish containing 15 ml Williams E medium (Invitrogen) supplemented with 10% (vol/vol) fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 11 mM glucose. The liver capsule was disrupted and the cells were dispersed. Damaged hepatocytes were removed by Percoll (Amersham Biosciences, Freiburg, Germany) gradient centrifugation. The cell density was adjusted to 4 x 105 cells/ml, and 1 x 105 cells/cm2 were allowed to attach to collagen-coated cell culture plates (BD Biosciences, Franklin Lakes, NJ).
2-Deoxy-D-[3H]glucose uptake in hepatocytes
Cells were washed and starved subsequently for 30 min with Williams E complete medium containing 2 mM glucose and then incubated in the same medium with 5 mM glucose and 5.6 μCi/ml of 2-deoxy-D-[3H]glucose (Amersham Biosciences) for 15 min, with or without test compound. The cells were then washed twice with ice-cold PBS containing 20 mM glucose and lysed with 1 M NaOH. Liquid scintillation was used to determine the amount of radioactivity in the lysates.
GK protein expression
INS-1E cells were cultured in the absence or presence of a GK activator for 32 h in the culture medium containing either 3 or 15 mM glucose. After incubation, cells were lysed in M-Per buffer (Pierce Biotechnology, Rockford, IL) supplemented with the protease inhibitor cocktail (Roche Diagnostics). The lysate was centrifuged at 1000 x g for 10 min; the supernatant was collected, and the protein concentration was measured. Twenty micrograms protein from each sample were applied to SDS-PAGE. Proteins were separated and transferred to polyvinyl difluoride membrane for immunoblotting with anti-GK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Membranes were also probed with anti-actin antibody (Sigma-Aldrich, Inc., St. Louis, MO) to demonstrate equal protein loading for the samples. A VersaDoc 3000 imaging system (Bio-Rad Laboratories, Hercules, CA) was used for capture and analysis of the enhanced chemiluminescent signal.
Oral glucose tolerance test (OGTT)
OGTT was performed in healthy conscious Wistar male rats (250–300 g, Harlan, Horst, The Netherlands). Rats were fasted overnight before performing the test. Test compound was given orally (formulation: 90% water, 10 acacia, 0.1% antifoam 1510), followed 2 h later by an oral glucose challenge (2 g/kg). Blood samples were collected from tail tip just before and after the glucose challenge (0, 15, 30, 60, 90, 120 min), and plasma glucose concentrations were determined.
Statistical analysis
Results are presented as mean values ± SEM for the indicated number of experiments. Statistical significances were evaluated using Student’s t test for paired data or Dunnett’s test for multiple comparisons. Curve fitting was performed with SigmaPlot 2000 (version 6 software; Systat Software Inc., Richmond, CA).
Results
In search of GK activators, we identified a novel enantiopure compound, 2-(S)-cyclohexyl-1-(R)-(4-methanesulfonyl-phenyl)-cyclopropanecarboxylic acid thiazol-2-ylamide (LY2121260; Fig. 1A, inset), which increases GK activity. The compound altered both the glucose affinity of the enzyme and the velocity of the reaction (Fig. 1A). LY2121260 (10 μM) increased maximal velocity by 40% and decreased glucose ligand concentration required for a half-maximal activity: control, 6.8 ± 0.5 mM; LY2121260, 0.4 ± 0.1 mM. Submicromolar concentrations of the compound were found to activate GK in the presence of both 2 and 10 mM glucose (Fig. 1B, at 2 mM glucose, EC50 = 0.73 ± 0.04 μM; 10 mM glucose, EC50 = 0.14 ± 0.01 μM). This activation of enzyme activity was found to be unique for GK because LY2121260 at concentrations up to 100 μM had no effect on the activity of human hexokinase I in an enzymatic assay analogous to the GK activity assay (data not shown). Interestingly, the opposite enantiomer of LY2121260 showed moderate activation of GK (at 10 mM glucose EC50 = 14.6 ± 0.5 μM, data not shown), demonstrating that the activator undergoes a stereospecific interaction with the enzyme.
To better understand the nature of the interaction between LY2121260 and GK, cocrystals of LY2121260 and the human islet GK isoform were grown in the presence of glucose, and a crystal structure of the resulting tertiary complex was obtained. The activator was found to be buried in a deep pocket in the hinge region of the enzyme remote from the active site in a manner reminiscent of that reported for other small molecule GK activators, suggesting that it activates the enzyme in a fashion similar to those compounds (12, 14) (Fig. 2A). The backbone hydrogen and carbonyl oxygen of Arg63 anchor the thiazole nitrogen and the amide NH of the activator, respectively, with hydrogen bonds (Fig. 2B). The compound cyclohexyl moiety is embedded in a lipophilic environment created by V62, M210, M235, and Y214 but also has a solvent accessible area toward E221. Recently a number of either naturally occurring or artificial GK-activating mutations have been characterized: V62M, T65I, W99R, Y214C, V455M, A456V (11, 21). Interestingly, these GK-activating mutations are located in the proximity of the GK activator binding site.
In pancreatic -cells, activation of GK results in enhanced glucose use and, ultimately, stimulation of insulin secretion (1). Incubation of rat pancreatic islets with different concentrations of LY2121260 produced a dose-dependent increase in insulin secretion at 10 mM glucose (Fig. 3A). The glucose dependency of the effects of 1 μM LY2121260 on insulin secretion was further studied (Fig. 3B). The activator was found to stimulate insulin secretion at intermediate glucose concentrations of 4 and 8 mM. Maximal stimulation of insulin secretion (4-fold) was observed at 8 mM glucose. At lower (2 mM) and higher (14 and 20 mM) glucose concentrations, the compound did not significantly alter the amount of secreted insulin. In addition to primary -cells, the GK activator stimulated insulin secretion in insulinoma cells. In INS-1E cells at 5 mM glucose, LY2121260 produced a 2-fold increase in insulin secretion (EC50 = 0.11 ± 0.02 μM, n = 4, data not shown).
In pancreatic -cells, GK protein levels are regulated by glucose on a posttranscriptional level (22). It has been suggested that glucose stabilizes GK via induction of large conformational changes in the enzyme, leading to increased protein levels (1). We examined the effect of LY2121260 on GK protein levels in INS-1E cells. In the absence of the activator, 32 h culture of INS-1E cells at 15 mM glucose resulted in an almost 3-fold increase in GK protein levels, compared with GK levels in cells cultured at 3 mM glucose (Fig. 4). In the presence of 3 mM glucose, 1 μM LY2121260 induced a 2-fold increase in GK protein levels. At 15 mM glucose, inclusion of the activator in the incubation medium resulted in a slight increase in GK protein levels.
In addition to its role in pancreatic -cells, GK plays an important role in the control of glucose metabolism in the liver. Liver GK activity determines the rates of glucose use and glycogen synthesis (3, 5). To determine the effect of the GK activator on hepatic glucose use, we measured 2-deoxy-D-[3H]glucose uptake in primary rat hepatocytes in the presence and absence of the activator (Fig. 5). LY2121260 enhanced 2-deoxy-D-[3H]glucose uptake in rat hepatocytes by 140% (EC50 = 1.7 ± 0.4 μM).
The ability of the GK activator to modulate blood glucose levels in vivo was studied in OGTT in healthy Wistar rats. LY2121260 (3–50 mg/kg) was administered to overnight-fasted rats 2 h before an oral glucose challenge (Fig. 6A). Treatment with the activator resulted in a dose-dependent decrease in fasting blood glucose levels before the glucose challenge. At the highest dose studied, the activator decreased the blood glucose concentration from 97 to 75 mg/dl (P < 0.05). After the glucose challenge, blood glucose levels rose rapidly. However, in animals treated with LY2121260, the rise in blood glucose levels was reduced dose dependently (Fig. 6A). Treatment with 50 mg/kg LY2121260 resulted in 28% reduction in glucose exposure, compared with control, as measured by the area under the curve for glucose (AUC glucose) (Fig. 6B).
Discussion
Blood glucose levels are tightly regulated in the body via multiple mechanisms, and a number of tissues are equipped with sensory systems that allow them to detect and respond to changes between fasting and fed glucose levels. It has been demonstrated that GK is the major glucose-sensing enzyme in a number of metabolically responsive organs: brain, liver, gut, and pancreas (23). In patients with type 2 diabetes, the control of glucose homeostasis is severely disturbed, leading to hyperglycemia. In both the diabetic liver and pancreas, the sensitivity to glucose is reduced. Insulin secretion from diabetic pancreatic islets in response to high glucose is severely impaired, whereas the response to other stimuli can be preserved (24, 25). In the liver, the ability of high glucose to suppress hepatic glucose production is diminished in diabetic patients (26). Due to its important role in controlling both glucose-induced insulin secretion and hepatic glucose production (1, 27), GK is an appealing target for the treatment of type 2 diabetes, and it is postulated that agents enhancing activity of the enzyme should improve glucose control in the disease state.
Recently, the discovery and development of small-molecule GK activators have been reported (12, 13, 14). The activators stimulate GK activity via binding to an allosteric site on the enzyme and have been shown to decrease plasma glucose levels in healthy and diabetic animals via stimulation of both insulin secretion and hepatic glucose metabolism. We demonstrate here that the novel GK activator LY2121260 promotes insulin secretion in pancreatic islets and stimulates glucose use in hepatocytes, thereby lowering blood glucose levels in vivo after an oral glucose challenge in healthy Wistar rats.
The novel GK activator LY2121260 increased both the affinity of the enzyme for glucose and the maximum velocity of the enzyme-catalyzed reaction. The GK activator induced a large decrease in ligand concentration required for a half-maximal activity for glucose, which leads to a shift in the set point for glucose use and insulin secretion. In the presence of the activator, glucose use in liver as well as insulin secretion in pancreas can be triggered at lower-than-usual glucose concentrations, potentially leading to hypoglycemia. In fact, treatment of healthy Wistar rats with LY2121260 led to a decrease in fasting blood glucose levels, presumably via increased glucose use and enhanced insulin secretion. On the other hand, the activator is 5-fold more potent in the enzymatic assay at 10 mM glucose than at 2 mM glucose. This dependency of the activator potency on glucose levels suggests that it should be possible to find optimal doses of the activator, at which activation occurs preferentially at elevated glucose levels. However, great care should be taken to develop GK activators devoid of hypoglycemia effects.
In pancreatic -cells, GK is the rate-limiting enzyme in glycolysis and, therefore, in glucose-induced insulin secretion (1). Stimulation of insulin secretion with the GK activator required a certain amount of glucose present in the incubation medium. At a subphysiological glucose concentration (2 mM), the activator did not significantly affect insulin secretion at concentrations up to 3 μM. However, at 4 mM glucose LY2121260 produced a significant increase in insulin secretion. The activator was more efficient in stimulation of insulin secretion at intermediate glucose concentrations (8–10 mM), shifting the dose-response curve for glucose to the left with no effect on the maximal increase in glucose-induced insulin secretion. The fact that the activator does not further increase insulin secretion stimulated by 20 mM glucose is consistent with the observation that at high glucose concentrations, other metabolic enzymes downstream of GK become rate limiting and insulin secretion no longer parallels GK activity (2, 28).
GK expression is finely regulated to adapt to changes in glucose flux at different metabolic states. Whereas expression of hepatic GK is regulated mainly by insulin and glucagon on the level of gene transcription (29), pancreatic GK expression is mainly regulated on a posttranscriptional level by glucose (22, 30). When glucose binds to GK, it induces large conformational changes in the enzyme. Furthermore, it has been demonstrated that the glucose-bound form of GK is more stable than the apo-enzyme to oxidation and degradation (1, 16). We observed an increase in GK protein levels in -cells cultured at elevated glucose concentrations. Interestingly, in -cells cultured at low glucose in the presence of LY2121260, GK protein levels were also increased, implying that binding of the activator maintains the enzyme in a more stable form, possibly by keeping it in a glucose-bound conformation (14). Thus, increases in GK activity resulting from chronic administration of a GK activator to -cells are likely the result of not just modified enzyme kinetics but also of increased quantities of enzyme.
In summary, we have discovered a novel small-molecule GK activator, 2-(S)-cyclohexyl-1-(R)-(4-methanesulfonyl-phenyl)-cyclopropanecarboxylic acid thiazol-2-ylamide (LY2121260), which activates GK in a fashion similar to other small molecule activators reported recently (12, 13, 14). We have demonstrated that LY2121260 enhances GK activity in a glucose-dependent manner in vitro; enhances insulin secretion in both isolated pancreatic islets and in INS-1E cells; and increases glucose use in rat hepatocytes. In addition, we have established that incubation of -cells with the GK activator results in elevated GK protein levels, suggesting that enhanced insulin secretion from -cells during chronic treatment with a GK activator is not due solely to increased enzymatic activity but rather also to elevated protein levels. These results taken together support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in so doing may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.
Acknowledgments
We thank Gina Tillmann, Dagmar Volk, Dirk Wesemann, Henrik Benjes, and Christina Schmidt for excellent technical assistance.
Footnotes
Abbreviations: AUC glucose, Area under the curve for glucose response; DTT, dithiothreitol; GK, glucokinase; OGTT, oral glucose tolerance test.
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Lilly Research Laboratories (S.L.B., J.D.D., H.G., Y.W.), Indianapolis, Indiana 46285
Lilly Development Centre (A.D.), 1348 Mont-Saint-Guibert, Belgium
Abstract
The glucose-sensing enzyme glucokinase (GK) plays a key role in glucose metabolism. We report here the effects of a novel glucokinase activator, LY2121260. The activator enhanced GK activity via binding to the allosteric site located in the hinge region of the enzyme. LY2121260 stimulated insulin secretion in a glucose-dependent manner in pancreatic -cells and increased glucose use in rat hepatocytes. In addition, incubation of -cells with the GK activator resulted in increased GK protein levels, suggesting that enhanced insulin secretion on chronic treatment with a GK activator may be due to not only changed enzyme kinetics but also elevated enzyme levels. Animals treated with LY2121260 showed an improved glucose tolerance after oral glucose challenge. These results support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in doing so may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.
Introduction
GLUCOKINASE (GK), A MEMBER of the hexokinase family, catalyzes the first step in glycolysis, phosphorylation of glucose to glucose 6-phosphate. It is a unique hexokinase in that it has a low affinity for glucose, demonstrates non-Michaelis-Menten kinetics (Hill coefficient is around 1.5) and displays no inhibition by the product of the reaction, glucose 6-phosphate (1). These particular features make GK an ideal sensor of physiological changes in blood glucose levels, translating them into changes in the metabolic status of the cell.
GK has a limited tissue distribution and is expressed predominantly in metabolically active tissues: brain, gut, liver, and pancreas. The roles of GK in pancreas and liver are well understood. In pancreatic -cells, GK is the rate-limiting enzyme in glucose metabolism and determines the rate of glucose-induced insulin secretion (1, 2). In liver, GK activity determines the rate of glucose use and glycogen synthesis (3). These roles make GK the critical player in the regulation of blood glucose levels. Activation of the enzyme at postprandial glucose levels produces a rise in insulin secretion from the pancreas as well as stimulation of glucose uptake and glycogen synthesis in liver, the combination of which leads to an increase in glucose disposal and the return of blood sugar to basal levels.
The significance of GK in the control of blood glucose is underscored by data from animal models, in which GK activity has been modulated, and in humans possessing GK mutations. GK-deficient mice die early after birth due to severe diabetes (4, 5). Pancreatic -cell or liver-specific GK knockout mice also display hyperglycemia, the severity of the phenotype being more pronounced in -cell knockouts (5). On the other hand, overexpression of GK leads to lower basal blood glucose levels and resistance to the development of high-fat-induced diabetes (6, 7). In humans, more than 150 naturally occurring mutations in GK have been reported to date (8). It has been shown that mutations in a single GK gene allele that impair enzyme function cause type 2 maturity onset diabetes of young (MODY2) (9), whereas inactivating mutations in both GK alleles cause permanent neonatal diabetes (10). In addition, GK mutations that increase enzyme activity cause hyperinsulinemic hypoglycemia (11).
Recently small molecules that activate GK via binding to an allosteric site on the enzyme have been discovered (12, 13, 14). Through their ability to enhance the activity of GK, such so-called GK activators hold promise as a novel and effective treatment for type 2 diabetes. Herein we describe the effects of a novel GK activator in pancreatic -cells and hepatocytes.
Materials and Methods
Glucokinase enzymatic assay
An enzymatic GK assay using purified recombinant human islet GK was used to evaluate the effects of potential small molecule activators. In the assay, GK catalyzes glucose phosphorylation in the presence of ATP. The product of this reaction, glucose-6-phosphate, is then oxidized by an excess of glucose-6-phosphate dehydrogenase to produce gluconate-6-phosphate with concomitant reduction of nicotinamide adenine dinucleotide (15). Production of reduced nicotinamide adenine dinucleotide results in an increase in fluorescence, which was used to monitor GK activity. Human islet GK was expressed in Escherichia coli as a (His)6-tagged fusion protein and was purified via metal chelate affinity chromatography (16). The assay was performed in 96-well plates in a final incubation volume of 100 μl. The incubation mixture consisted of (in millimoles) 50 HEPES, 100 KCl, 5 MgCl2, 5 dithiothreitol (DTT), 10 Mg-ATP, 1 nicotinamide adenine dinucleotide (pH 7.4), 4 U/ml glucose-6-phosphate dehydrogenase, 100 ng/ml GK, and glucose at different concentrations.
GK crystal structure
Human GK was expressed as a glutathione-S-transferase fusion protein in E. coli and purified by affinity chromatography, followed by Factor Xa digestion to remove the glutathione-S-transferase and final gel filtration chromatography. Compound LY2121260 was added to GK protein solution (12 mg/ml) containing 20 mM HEPES (pH 7.6), 50 mM NaCl, 10 mM DTT, 50 mM glucose, and 1–2 mM DTT before crystallization. Crystals were grown at 4 C by the hanging drop method under the reservoir condition 0.1 M sodium citrate (pH 5.4), 20% polyethylene glycol 3350, and 0.2 M NH4I. Crystals belong to the space group P4(1)2(1)2 with unit cell parameters a = 70.43 , b = 70.43 , c = 188.75 . The diffraction data (resolution of 2.0 , Rmerge = 0.124, completeness of 96.5%) were collected using a Rigaku RU300 generator and an R-AXIS IV detector (Rigaku/MSC, Inc., The Woodlands, TX). The diffraction data were processed using HKL2000 (HKL Research, Inc., Charlottesville, VA). The crystal structure was determined by the method of molecular replacement (amore from the CCP4 Suite) (17) using coordinates from a previously determined GK complex (Wang, Y., unpublished results), which was determined by molecular replacement with the program CNX2000 (Accelrys, Inc., San Diego, CA) using the human brain hexokinase I structure 1HKB (18) as a search model. The program suite QUANTA 2000 (Accelrys) was used for model building and refinement was carried out with the program CNX2000.
Preparation and culture of pancreatic -cells
Rat pancreatic islets were isolated from male Wistar rats (250–300 g, Charles River Laboratories, Sulzfeld, Germany) as previously described (19). Islets were incubated in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum, 100 IU/ml penicillin, and 100 μg/ml streptomycin.
INS-1E cells were cultured in RPMI 1640 supplemented with 5% (vol/vol) heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin as described (20).
Insulin secretion assay
Insulin secretion in rat pancreatic islets was performed as described previously (19). For measurements of insulin secretion in INS-1E cells, cells were plated in 96-well plate at density of 20,000 cells/well and cultured for 2 d. Cells were preincubated at 37 C for 30 min in Earle’s balanced salt solution containing 0.1% BSA and no glucose. After preincubation cells were cultured for 1 h in the same buffer containing 5 mM glucose and different concentrations of the tested molecule. Insulin concentration in the collected supernatant was measured with ELISA using rat insulin antibody (Linco Research, St. Charles, MO).
Isolation and culture of hepatocytes
Hepatocytes were isolated from overnight-fasted male Wistar rats by a two-step in situ liver perfusion. Rats were anesthetized by 60 mg/kg pentobarbital. The hepatic portal vein was cannulated, and in a first perfusion step, the liver was flushed with 30 ml/min liver perfusion medium (Invitrogen) for 6 min. In a second step, the liver was digested by perfusion with liver perfusion medium supplemented with 5 mM NaCl, 5 mM CaCl2, and 23.3 mg/liter Liberase Blendzyme 3 (Roche Diagnostics, Mannheim, Germany) at a rate of 15 ml/min for approximately 9 min. The liver was then dissected and transferred to a dish containing 15 ml Williams E medium (Invitrogen) supplemented with 10% (vol/vol) fetal calf serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 11 mM glucose. The liver capsule was disrupted and the cells were dispersed. Damaged hepatocytes were removed by Percoll (Amersham Biosciences, Freiburg, Germany) gradient centrifugation. The cell density was adjusted to 4 x 105 cells/ml, and 1 x 105 cells/cm2 were allowed to attach to collagen-coated cell culture plates (BD Biosciences, Franklin Lakes, NJ).
2-Deoxy-D-[3H]glucose uptake in hepatocytes
Cells were washed and starved subsequently for 30 min with Williams E complete medium containing 2 mM glucose and then incubated in the same medium with 5 mM glucose and 5.6 μCi/ml of 2-deoxy-D-[3H]glucose (Amersham Biosciences) for 15 min, with or without test compound. The cells were then washed twice with ice-cold PBS containing 20 mM glucose and lysed with 1 M NaOH. Liquid scintillation was used to determine the amount of radioactivity in the lysates.
GK protein expression
INS-1E cells were cultured in the absence or presence of a GK activator for 32 h in the culture medium containing either 3 or 15 mM glucose. After incubation, cells were lysed in M-Per buffer (Pierce Biotechnology, Rockford, IL) supplemented with the protease inhibitor cocktail (Roche Diagnostics). The lysate was centrifuged at 1000 x g for 10 min; the supernatant was collected, and the protein concentration was measured. Twenty micrograms protein from each sample were applied to SDS-PAGE. Proteins were separated and transferred to polyvinyl difluoride membrane for immunoblotting with anti-GK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Membranes were also probed with anti-actin antibody (Sigma-Aldrich, Inc., St. Louis, MO) to demonstrate equal protein loading for the samples. A VersaDoc 3000 imaging system (Bio-Rad Laboratories, Hercules, CA) was used for capture and analysis of the enhanced chemiluminescent signal.
Oral glucose tolerance test (OGTT)
OGTT was performed in healthy conscious Wistar male rats (250–300 g, Harlan, Horst, The Netherlands). Rats were fasted overnight before performing the test. Test compound was given orally (formulation: 90% water, 10 acacia, 0.1% antifoam 1510), followed 2 h later by an oral glucose challenge (2 g/kg). Blood samples were collected from tail tip just before and after the glucose challenge (0, 15, 30, 60, 90, 120 min), and plasma glucose concentrations were determined.
Statistical analysis
Results are presented as mean values ± SEM for the indicated number of experiments. Statistical significances were evaluated using Student’s t test for paired data or Dunnett’s test for multiple comparisons. Curve fitting was performed with SigmaPlot 2000 (version 6 software; Systat Software Inc., Richmond, CA).
Results
In search of GK activators, we identified a novel enantiopure compound, 2-(S)-cyclohexyl-1-(R)-(4-methanesulfonyl-phenyl)-cyclopropanecarboxylic acid thiazol-2-ylamide (LY2121260; Fig. 1A, inset), which increases GK activity. The compound altered both the glucose affinity of the enzyme and the velocity of the reaction (Fig. 1A). LY2121260 (10 μM) increased maximal velocity by 40% and decreased glucose ligand concentration required for a half-maximal activity: control, 6.8 ± 0.5 mM; LY2121260, 0.4 ± 0.1 mM. Submicromolar concentrations of the compound were found to activate GK in the presence of both 2 and 10 mM glucose (Fig. 1B, at 2 mM glucose, EC50 = 0.73 ± 0.04 μM; 10 mM glucose, EC50 = 0.14 ± 0.01 μM). This activation of enzyme activity was found to be unique for GK because LY2121260 at concentrations up to 100 μM had no effect on the activity of human hexokinase I in an enzymatic assay analogous to the GK activity assay (data not shown). Interestingly, the opposite enantiomer of LY2121260 showed moderate activation of GK (at 10 mM glucose EC50 = 14.6 ± 0.5 μM, data not shown), demonstrating that the activator undergoes a stereospecific interaction with the enzyme.
To better understand the nature of the interaction between LY2121260 and GK, cocrystals of LY2121260 and the human islet GK isoform were grown in the presence of glucose, and a crystal structure of the resulting tertiary complex was obtained. The activator was found to be buried in a deep pocket in the hinge region of the enzyme remote from the active site in a manner reminiscent of that reported for other small molecule GK activators, suggesting that it activates the enzyme in a fashion similar to those compounds (12, 14) (Fig. 2A). The backbone hydrogen and carbonyl oxygen of Arg63 anchor the thiazole nitrogen and the amide NH of the activator, respectively, with hydrogen bonds (Fig. 2B). The compound cyclohexyl moiety is embedded in a lipophilic environment created by V62, M210, M235, and Y214 but also has a solvent accessible area toward E221. Recently a number of either naturally occurring or artificial GK-activating mutations have been characterized: V62M, T65I, W99R, Y214C, V455M, A456V (11, 21). Interestingly, these GK-activating mutations are located in the proximity of the GK activator binding site.
In pancreatic -cells, activation of GK results in enhanced glucose use and, ultimately, stimulation of insulin secretion (1). Incubation of rat pancreatic islets with different concentrations of LY2121260 produced a dose-dependent increase in insulin secretion at 10 mM glucose (Fig. 3A). The glucose dependency of the effects of 1 μM LY2121260 on insulin secretion was further studied (Fig. 3B). The activator was found to stimulate insulin secretion at intermediate glucose concentrations of 4 and 8 mM. Maximal stimulation of insulin secretion (4-fold) was observed at 8 mM glucose. At lower (2 mM) and higher (14 and 20 mM) glucose concentrations, the compound did not significantly alter the amount of secreted insulin. In addition to primary -cells, the GK activator stimulated insulin secretion in insulinoma cells. In INS-1E cells at 5 mM glucose, LY2121260 produced a 2-fold increase in insulin secretion (EC50 = 0.11 ± 0.02 μM, n = 4, data not shown).
In pancreatic -cells, GK protein levels are regulated by glucose on a posttranscriptional level (22). It has been suggested that glucose stabilizes GK via induction of large conformational changes in the enzyme, leading to increased protein levels (1). We examined the effect of LY2121260 on GK protein levels in INS-1E cells. In the absence of the activator, 32 h culture of INS-1E cells at 15 mM glucose resulted in an almost 3-fold increase in GK protein levels, compared with GK levels in cells cultured at 3 mM glucose (Fig. 4). In the presence of 3 mM glucose, 1 μM LY2121260 induced a 2-fold increase in GK protein levels. At 15 mM glucose, inclusion of the activator in the incubation medium resulted in a slight increase in GK protein levels.
In addition to its role in pancreatic -cells, GK plays an important role in the control of glucose metabolism in the liver. Liver GK activity determines the rates of glucose use and glycogen synthesis (3, 5). To determine the effect of the GK activator on hepatic glucose use, we measured 2-deoxy-D-[3H]glucose uptake in primary rat hepatocytes in the presence and absence of the activator (Fig. 5). LY2121260 enhanced 2-deoxy-D-[3H]glucose uptake in rat hepatocytes by 140% (EC50 = 1.7 ± 0.4 μM).
The ability of the GK activator to modulate blood glucose levels in vivo was studied in OGTT in healthy Wistar rats. LY2121260 (3–50 mg/kg) was administered to overnight-fasted rats 2 h before an oral glucose challenge (Fig. 6A). Treatment with the activator resulted in a dose-dependent decrease in fasting blood glucose levels before the glucose challenge. At the highest dose studied, the activator decreased the blood glucose concentration from 97 to 75 mg/dl (P < 0.05). After the glucose challenge, blood glucose levels rose rapidly. However, in animals treated with LY2121260, the rise in blood glucose levels was reduced dose dependently (Fig. 6A). Treatment with 50 mg/kg LY2121260 resulted in 28% reduction in glucose exposure, compared with control, as measured by the area under the curve for glucose (AUC glucose) (Fig. 6B).
Discussion
Blood glucose levels are tightly regulated in the body via multiple mechanisms, and a number of tissues are equipped with sensory systems that allow them to detect and respond to changes between fasting and fed glucose levels. It has been demonstrated that GK is the major glucose-sensing enzyme in a number of metabolically responsive organs: brain, liver, gut, and pancreas (23). In patients with type 2 diabetes, the control of glucose homeostasis is severely disturbed, leading to hyperglycemia. In both the diabetic liver and pancreas, the sensitivity to glucose is reduced. Insulin secretion from diabetic pancreatic islets in response to high glucose is severely impaired, whereas the response to other stimuli can be preserved (24, 25). In the liver, the ability of high glucose to suppress hepatic glucose production is diminished in diabetic patients (26). Due to its important role in controlling both glucose-induced insulin secretion and hepatic glucose production (1, 27), GK is an appealing target for the treatment of type 2 diabetes, and it is postulated that agents enhancing activity of the enzyme should improve glucose control in the disease state.
Recently, the discovery and development of small-molecule GK activators have been reported (12, 13, 14). The activators stimulate GK activity via binding to an allosteric site on the enzyme and have been shown to decrease plasma glucose levels in healthy and diabetic animals via stimulation of both insulin secretion and hepatic glucose metabolism. We demonstrate here that the novel GK activator LY2121260 promotes insulin secretion in pancreatic islets and stimulates glucose use in hepatocytes, thereby lowering blood glucose levels in vivo after an oral glucose challenge in healthy Wistar rats.
The novel GK activator LY2121260 increased both the affinity of the enzyme for glucose and the maximum velocity of the enzyme-catalyzed reaction. The GK activator induced a large decrease in ligand concentration required for a half-maximal activity for glucose, which leads to a shift in the set point for glucose use and insulin secretion. In the presence of the activator, glucose use in liver as well as insulin secretion in pancreas can be triggered at lower-than-usual glucose concentrations, potentially leading to hypoglycemia. In fact, treatment of healthy Wistar rats with LY2121260 led to a decrease in fasting blood glucose levels, presumably via increased glucose use and enhanced insulin secretion. On the other hand, the activator is 5-fold more potent in the enzymatic assay at 10 mM glucose than at 2 mM glucose. This dependency of the activator potency on glucose levels suggests that it should be possible to find optimal doses of the activator, at which activation occurs preferentially at elevated glucose levels. However, great care should be taken to develop GK activators devoid of hypoglycemia effects.
In pancreatic -cells, GK is the rate-limiting enzyme in glycolysis and, therefore, in glucose-induced insulin secretion (1). Stimulation of insulin secretion with the GK activator required a certain amount of glucose present in the incubation medium. At a subphysiological glucose concentration (2 mM), the activator did not significantly affect insulin secretion at concentrations up to 3 μM. However, at 4 mM glucose LY2121260 produced a significant increase in insulin secretion. The activator was more efficient in stimulation of insulin secretion at intermediate glucose concentrations (8–10 mM), shifting the dose-response curve for glucose to the left with no effect on the maximal increase in glucose-induced insulin secretion. The fact that the activator does not further increase insulin secretion stimulated by 20 mM glucose is consistent with the observation that at high glucose concentrations, other metabolic enzymes downstream of GK become rate limiting and insulin secretion no longer parallels GK activity (2, 28).
GK expression is finely regulated to adapt to changes in glucose flux at different metabolic states. Whereas expression of hepatic GK is regulated mainly by insulin and glucagon on the level of gene transcription (29), pancreatic GK expression is mainly regulated on a posttranscriptional level by glucose (22, 30). When glucose binds to GK, it induces large conformational changes in the enzyme. Furthermore, it has been demonstrated that the glucose-bound form of GK is more stable than the apo-enzyme to oxidation and degradation (1, 16). We observed an increase in GK protein levels in -cells cultured at elevated glucose concentrations. Interestingly, in -cells cultured at low glucose in the presence of LY2121260, GK protein levels were also increased, implying that binding of the activator maintains the enzyme in a more stable form, possibly by keeping it in a glucose-bound conformation (14). Thus, increases in GK activity resulting from chronic administration of a GK activator to -cells are likely the result of not just modified enzyme kinetics but also of increased quantities of enzyme.
In summary, we have discovered a novel small-molecule GK activator, 2-(S)-cyclohexyl-1-(R)-(4-methanesulfonyl-phenyl)-cyclopropanecarboxylic acid thiazol-2-ylamide (LY2121260), which activates GK in a fashion similar to other small molecule activators reported recently (12, 13, 14). We have demonstrated that LY2121260 enhances GK activity in a glucose-dependent manner in vitro; enhances insulin secretion in both isolated pancreatic islets and in INS-1E cells; and increases glucose use in rat hepatocytes. In addition, we have established that incubation of -cells with the GK activator results in elevated GK protein levels, suggesting that enhanced insulin secretion from -cells during chronic treatment with a GK activator is not due solely to increased enzymatic activity but rather also to elevated protein levels. These results taken together support the concept that GK activators represent a new class of compounds that increase both insulin secretion and hepatic glucose use and in so doing may prove to be effective agents for the control of blood glucose levels in patients with type 2 diabetes.
Acknowledgments
We thank Gina Tillmann, Dagmar Volk, Dirk Wesemann, Henrik Benjes, and Christina Schmidt for excellent technical assistance.
Footnotes
Abbreviations: AUC glucose, Area under the curve for glucose response; DTT, dithiothreitol; GK, glucokinase; OGTT, oral glucose tolerance test.
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