Glucokinase as glucose sensor and metabolic signal generator in pancreatic beta-cells and hepatocytes

This article reviews evidence for a pivotal role of glucokinase as glucose sensor of the pancreatic beta-cells. Glucokinase explains the capacity, hexose specificity, affinities, sigmoidicity, and anomeric preference of pancreatic islet glycolysis, and because stimulation of glucose metabolism is a prerequisite of glucose stimulation of insulin release, glucokinase also explains many characteristics of this beta-cell function. Glucokinase of the beta-cell is induced or activated by glucose in contrast to liver glucokinase, which is regulated by insulin. Tissue-specific regulation corresponds with observations that liver and pancreatic beta-cell glucokinase are structurally distinct. Glucokinase could play a glucose-sensor role in hepatocytes as well, and certain forms of diabetes mellitus might be due to glucokinase deficiencies in pancreatic beta-cells, hepatocytes, or both.     

Insights into the biochemical and genetic basis of glucokinase activation from naturally occurring hypoglycemia mutations

Glucokinase (GCK) is a key regulatory enzyme in the pancreatic beta-cell and catalyzes the rate-limiting step for beta-cell glucose metabolism. We report two novel GCK mutations (T65I and W99R) that have arisen de novo in two families with familial hypoglycemia. Insulin levels, although inappropriately high for the degree of hypoglycemia, remain regulated by fluctuations in glycemia, and pancreatic histology was normal. These mutations are within the recently identified heterotropic allosteric activator site in the theoretical model of human beta-cell glucokinase. Functional analysis of the purified recombinant glutathionyl S-transferase fusion proteins of T65I and W99R GCK revealed that the kinetic changes result in a relative increased activity index (a measure of the enzyme's phosphorylating potential) of 9.81 and 6.36, respectively, compared with wild-type. The predicted thresholds for glucose-stimulated insulin release using mathematical modeling were 3.1 (T65I) and 2.8 (W99R) mmol/l, which were in line with the patients' fasting glucose. In conclusion, we have identified two novel spontaneous GCK-activating mutations whose clinical phenotype clearly differs from mutations in ATP-sensitive K(+) channel genes. In vitro studies confirm the validity of structural and functional models of GCK and the putative allosteric activator site, which is a potential drug target for the treatment of type 2 diabetes.     

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells

We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.     

Identification of glucokinase as an alloxan-sensitive glucose sensor of the pancreatic beta-cell

Alloxan inactivated glucokinase in intact, isolated pancreatic islets incubated in vitro. Inactivation of glucokinase was antagonized by 30 mM glucose present during incubation of islets with alloxan. Glucokinase partially purified from transplantable insulinomas or rat liver was inactivated by alloxan with a half-maximal effect at 2-4 microM alloxan. Inactivation of purified glucokinase was antagonized by glucose, mannose, and 2-deoxyglucose in order of decreasing potency but not by 3-O-methylglucose. Glucose anomers at 6 and 14 mM were discriminated as protecting agents, with the alpha-anomer more effective than the beta-anomer. Glucokinase was not protected from alloxan inactivation by N-acetylglucosamine, indicating that the reactive site for alloxan is not the active site; therefore, glucose may protect glucokinase by inducing a conformational change. Glucokinase is thought to be the glucose sensor of the pancreatic beta-cell. The finding that glucokinase is inactivated by alloxan and protected by glucose with discrimination of its anomers similar to inhibition of glucose-stimulated insulin secretion by alloxan supports this hypothesis and appears to explain the mechanism for inhibition of hexose-stimulated insulin secretion by this agent and the unique role of glucose and mannose as protecting agents.     

Novel insights into the regulation of the bound and diffusible glucokinase in MIN6 beta-cells

A stable MIN6 beta-cell clone overexpressing glucokinase as an enhanced cyan fluorescent protein (ECFP) fusion construct was generated for analysis of glucokinase regulation in these glucose-responsive insulin-secreting cells. A higher glucokinase enzyme activity accompanied by an improved glucose-induced insulin secretion indicated the integration of ECFP-glucokinase into the functional pool of glucokinase protein in MIN6-ECFP-glucokinase cells. Fluorescence recovery after photobleaching experiments of MIN6-ECFP-glucokinase cells and photoactivation of a transiently transfected photoswitchable cyan fluorescent protein (PS-CFP)-glucokinase construct in MIN6 cells indicate a higher motility of the diffusible glucokinase fraction at high glucose concentrations. In agreement with previous studies, we observed significant binding of ECFP-glucokinase to insulin secretory granules. Using fluorescence lifetime imaging, we obtained evidence for an association between glucokinase and alpha-tubulin in MIN6-ECFP-glucokinase cells. Furthermore, immunohistochemistry and fluorescence resonance energy transfer analysis by acceptor photobleaching showed distinct association between endogenous glucokinase and alpha-tubulin as well as beta-tubulin in MIN6 cells. Interestingly, glucokinase was also colocalized with kinesin, a motor protein involved in insulin secretory granule movement. Therefore, we suggest a role of a bound glucokinase protein fraction in the regulation of insulin granule movement along tubulin filaments.     

Activation of acetyl-CoA carboxylase by a glutamate- and magnesium-sensitive protein phosphatase in the islet beta-cell.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of long-chain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)-dephosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet beta-cell. ACC was quantitated in normal rat islets, human islets, and clonal beta-cells (HIT-15 or INS-1) using a [(14)C]bicarbonate fixation assay. In the beta-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations (<50 nmol/l) that inhibited protein phosphatase 2A (PP2A). Furthermore, pretreatment of the cytosolic fraction with anti-PP2A serum attenuated the glutamate- and magnesium-mediated activation of ACC, thereby suggesting that ACC may be regulated by an OKA-sensitive PP2A-like enzyme. Streptavidin-agarose chromatography studies have indicated that glutamate- and magnesium-mediated effects on ACC are attributable to activation of ACC's dephosphorylation; this suggests that the stimulatory effects of glutamate and magnesium on ACC might involve activation of an OKA-sensitive PP2A-like enzyme that dephosphorylates and activates ACC. In our study, 5-amino-imidazolecarboxamide (AICA) riboside, a stimulator of AMP kinase, significantly inhibited glucose-mediated activation of ACC and insulin secretion from isolated beta-cells. Together, our data provide evidence for a unique regulatory mechanism for the activation of ACC in the pancreatic beta-cell, leading to the generation of physiological signals that may be relevant for physiological insulin secretion.     

Control of glucose phosphorylation and glucose usage in clonal insulinoma cells

Glucose metabolism was investigated in two established clonal insulinoma cell lines (RINm5F and HIT) and in a newly developed line of mouse insulinoma cells (IgSV195). The hexokinase capacity in the homogenates of RINm5F cells was 22.1 +/- 3.23 U/g protein, but glucokinase was barely detectable (0.06 +/- 0.013 U/g protein). In contrast, both HIT and IgSV195 cells contained glucokinase (1.5 +/- 0.17 and 1.0 +/- 0.16 U/g protein, respectively) in addition to hexokinase activity. Glucose usage by the intact cells qualitatively reflected the glucose phosphorylation found in the cell-free extracts. RINm5F cells exhibited a high glucose usage rate with one high-affinity component, whereas both HIT and IgSV195 cells showed two components with different glucose affinities. HIT and IgSV195 cells may be useful for a model of pancreatic beta-cell glycolysis.     

Peroxisome proliferator-activated receptor-gamma regulates expression of PDX-1 and NKX6.1 in INS-1 cells

In the 60% pancreatectomy (Px) rat model of beta-cell adaptation, normoglycemia is maintained by an initial week of beta-cell hyperplasia that ceases and is followed by enhanced beta-cell function. It is unknown how this complex series of events is regulated. We studied isolated islets and pancreas sections from 14-day post-Px versus sham-operated rats and observed a doubling of beta-cell nuclear peroxisome proliferator-activated receptor (PPAR)-gamma protein, along with a 2-fold increase in nuclear pancreatic duodenal homeobox (Pdx)-1 protein and a 1.4-fold increase in beta-cell nuclear Nkx6.1 immunostaining. As PPAR-gamma activation is known to both lower proliferation and have prodifferentiation effects in many tissues, we studied PPAR-gamma actions in INS-1 cells. A 3-day incubation with the PPAR-gamma agonist troglitazone reduced proliferation and increased Pdx-1 and Nkx6.1 immunostaining, along with glucokinase and GLUT2. Also, a 75% knockdown of PPAR-gamma using RNA interference lowered the mRNA levels of Pdx-1, glucokinase, GLUT2, and proinsulin II by more than half. Our results show a dual effect of PPAR-gamma in INS-1 cells: to curtail proliferation and promote maturation, the latter via enhanced expression of Pdx-1 and Nkx6.1. Additional studies are needed to determine whether there is a regulatory role for PPAR-gamma signaling in the beta-cell adaptation following a 60% Px in rats.     

Interaction of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) with glucokinase activates glucose phosphorylation and glucose metabolism in insulin-producing cells

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) was recently identified as a new intracellular binding partner for glucokinase (GK). Therefore, we studied the importance of this interaction for the activity status of GK and glucose metabolism in insulin-producing cells by overexpression of the rat liver and pancreatic islet isoforms of PFK-2/FBPase-2. PFK-2/FBPase-2 overexpression in RINm5F-GK cells significantly increased the GK activity by 78% in cells expressing the islet isoform, by 130% in cells expressing the liver isoform, and by 116% in cells expressing a cAMP-insensitive liver S32A/H258A double mutant isoform. Only in cells overexpressing the wild-type liver PFK-2/FBPase-2 isoform was the increase of GK activity abolished by forskolin, apparently due to the regulatory site for phosphorylation by a cAMP-dependent protein kinase. In cells overexpressing any isoform of the PFK-2/FBPase-2, the increase of the GK enzyme activity was antagonized by treatment with anti-FBPase-2 antibody. Increasing the glucose concentration from 2 to 10 mmol/l had a significant stimulatory effect on the GK activity in RINm5F-GK cells overexpressing any isoform of PFK-2/FBPase-2. The interaction of GK with PFK-2/FBPase-2 takes place at glucose concentrations that are physiologically relevant for the activation of GK and the regulation of glucose-induced insulin secretion. This new mechanism of posttranslational GK regulation may also represent a new site for pharmacotherapeutic intervention in type 2 diabetes treatment.     

Severe persistent hyperinsulinemic hypoglycemia due to a de novo glucokinase mutation

Glucokinase (GK) is a glycolytic key enzyme that functions as a glucose sensor in the pancreatic beta-cell, where it governs glucose-stimulated insulin secretion (GSIS). Heterozygous inactivating mutations in the glucokinase gene (GCK) cause a mild form of diabetes (maturity-onset diabetes of the young [MODY]2), and activating mutations have been associated with a mild form of familial hyperinsulinemic hypoglycemia. We describe the first case of severe persistent hyperinsulinemic hypoglycemia due to a "de novo" mutation in GCK (Y214C). A baby girl presented with hypoglycemic seizures since the first postnatal day as well as with inappropriate hyperinsulinemia. Severe hypoglycemia persisted even after treatment with diazoxide and subtotal pancreatectomy, leading to irreversible brain damage. Pancreatic histology revealed abnormally large and hyperfunctional islets. The mutation is located in the putative allosteric activator domain of the protein. Functional studies of purified recombinant glutathionyl S-transferase fusion protein of GK-Y214C showed a sixfold increase in its affinity for glucose, a lowered cooperativity, and increased kcat. The relative activity index of GK-Y214C was 130, and the threshold for GSIS predicted by mathematical modeling was 0.8 mmol/l, compared with 5 mmol/l in the wild-type enzyme. In conclusion, we have identified a de novo GCK activating mutation that causes hyperinsulinemic hypoglycemia of exceptional severity. These findings demonstrate that the range of the clinical phenotype caused by GCK mutations varies from complete insulin deficiency to extreme hyperinsulinemia.     

From clinicogenetic studies of maturity-onset diabetes of the young to unraveling complex mechanisms of glucokinase regulation

Glucokinase functions as a glucose sensor in pancreatic beta-cells and regulates hepatic glucose metabolism. A total of 83 probands were referred for a diagnostic screening of mutations in the glucokinase (GCK) gene. We found 11 different mutations (V62A, G72R, L146R, A208T, M210K, Y215X, S263P, E339G, R377C, S453L, and IVS5 + 1G>C) in 14 probands. Functional characterization of recombinant glutathionyl S-transferase-G72R glucokinase showed slightly increased activity, whereas S263P and G264S had near-normal activity. The other point mutations were inactivating. S263P showed marked thermal instability, whereas the stability of G72R and G264S differed only slightly from that of wild type. G72R and M210K did not respond to an allosteric glucokinase activator (GKA) or the hepatic glucokinase regulatory protein (GKRP). Mutation analysis of the role of glycine at position 72 by substituting E, F, K, M, S, or Q showed that G is unique since all these mutants had very low or no activity and were refractory to GKRP and GKA. Structural analysis provided plausible explanations for the drug resistance of G72R and M210K. Our study provides further evidence that protein instability in combination with loss of control by a putative endogenous activator and GKRP could be involved in the development of hyperglycemia in maturity-onset diabetes of the young, type 2. Furthermore, based on data obtained on G264S, we propose that other and still unknown mechanisms participate in the regulation of glucokinase.     

Nuclear protein p8 is associated with glucose-induced pancreatic beta-cell growth

On its own, glucose is a major factor for proliferation of pancreatic beta-cells and is also an essential prerequisite for IGF-I and growth hormone-induced growth of these cells. p8 was originally identified as an emergency gene product upregulated in pancreatic acinar cells in response to acute pancreatitis. p8 was further shown to be involved in a broad range of biological functions, including cell growth, growth arrest, apoptosis, and tumor development. These in part opposite actions may be related to distinct stimuli and pathways in certain conditions and cell types. Here we demonstrate that p8 is widely expressed in human pancreatic islets in vivo and in several beta-cell lines in vitro. Based on this observation, we tested the hypothesis that p8 production in pancreatic beta-cells is regulated by glucose. Incubation of rat INS-1 beta-cells with 25 mmol/l glucose resulted in a continuous increase of proliferating cell numbers. This was accompanied by a strong upregulation of p8 mRNA and protein expression, indicating that p8 is a physiological mediator of glucose-induced pancreatic beta-cell growth. Binding of glucose-activated protein kinase C (PKC) to two PKC sites within a highly conserved region of the p8 protein may be a possible mechanism linking glucose and p8 pathways leading to proliferation.     

Insulin receptor-related receptor is expressed in pancreatic beta-cells and stimulates tyrosine phosphorylation of insulin receptor substrate-1 and -2.

The receptor-type protein tyrosine kinases in murine pancreatic islets were screened to identify possible growth/differentiation factors in pancreatic beta-cells. The analysis revealed that insulin receptor-related receptor (IRR) is highly expressed in the islets as well as in several highly differentiated beta-cell lines derived from transgenic mice. Islets predominantly contain IRR as uncleaved proreceptors compared with IRR as processed forms in the beta-cell lines, suggesting that the activity of IRR is regulated on the level of processing proteases in vivo. To examine the IRR signaling pathway, a chimeric receptor consisting of the extracellular domain of insulin receptor and the intracellular domain of IRR was expressed in Chinese hamster ovary cells. The hybrid receptor is functional because insulin is capable of tyrosine-phosphorylating the catalytic domain in these cells. It also stimulates the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2, indicating that both proteins serve as substrates of IRR-protein tyrosine kinase in intact cells. The phenotype of the IRS-2 knockout mouse recently reported suggests that an IRS-2-mediated signaling pathway controls the compensatory increase in pancreatic beta-cell mass in insulin-resistant states. From our findings of the specific expression of IRR and its ability of signaling to IRS-2, we speculate that this receptor might play a role in the regulation of beta-cell mass.     

An adenovirus vector for efficient RNA interference-mediated suppression of target genes in insulinoma cells and pancreatic islets of langerhans

Silencing gene expression by RNA interference (RNAi) can provide insight into gene function but requires efficient delivery of small interfering RNAs (siRNAs) into cells. Introduction of exogenous nucleic acids can be especially difficult in cultured pancreatic islets. This article describes a method for making recombinant adenoviruses that efficiently drive expression of siRNAs in islet beta-cells and a beta-cell-derived cell line. Transduction with a virus expressing an siRNA specific for GLUT2 reduced GLUT2 mRNA and protein levels by 80% in the INS-1-derived beta-cell line, 832/13, and GLUT2 protein levels by >90% in primary rat islets. Another virus expressing an siRNA specific for glucokinase (GK) caused 80% suppression of GK mRNA and 50% suppression of GK protein levels in 832/13 cells. These experiments validate recombinant adenoviral RNAi vectors as a useful tool for suppression of the expression of specific genes in pancreatic islets and beta-cell lines. Advantages of this approach include 1) the high efficiency of adenovirus-mediated gene transfer in insulinoma cell lines and rat islets and 2) the rapidity with which RNAi constructs can be prepared and tested relative to stable-transfection strategies.     

Pancreatic-derived factor (FAM3B), a novel islet cytokine,induces apoptosis of insulin-secreting beta-cells

PANDER (PANcreatic DERived factor, FAM3B), a newly discovered secreted cytokine, is specifically expressed at high levels in the islets of Langerhans of the endocrine pancreas. To evaluate the role of PANDER in beta-cell function, we investigated the effects of PANDER on rat, mouse, and human pancreatic islets; the beta-TC3 cell line; and the alpha-TC cell line. PANDER protein was present in alpha- and beta-cells of pancreatic islets, insulin-secreting beta-TC3 cells, and glucagon-secreting alpha-TC cells. PANDER induced islet cell death in rat and human islets. Culture of beta-TC3 cells with recombinant PANDER had a dose-dependent inhibitory effect on cell viability. This effect was also time-dependent. PANDER caused apoptosis of beta-cells as assessed by electron microscopy, annexin V fluorescent staining, and flow-cytometric terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. PANDER did not affect cytosolic Ca(2+) levels or nitric oxide levels. However, PANDER activated caspase-3. Hence, PANDER may have a role in the process of pancreatic beta-cell apoptosis.     

Concordant glucose induction of glucokinase, glucose usage, and glucose-stimulated insulin release in pancreatic islets maintained in organ culture.

Using cultured islets as the experimental system, this study established dosage-response and time-dependency curves of the inductive glucose effect on glucose-stimulated insulin release, glucose usage, and glucokinase activity. Glucose-stimulated insulin release in islets cultured for 1, 2, or 7 days was increased as a function of glucose concentration in the culture medium and as a function of time. Glucose usage in the cultured islets showed a close relationship with glucose concentration in the culture medium at both 2 and 7 days of culture. Glucokinase activity increased in islets cultured for 1, 2, or 7 days as a function of increasing glucose concentrations in the culture medium and as a function of time. The V(max) of glucokinase in islets cultured for 7 days in medium containing 30 mM glucose was twice the value of freshly isolated islets and was almost fivefold higher than that in islets cultured for 7 days in 3 mM glucose. The glucose induction of glucose-stimulated insulin release, of glucose usage, and of glucokinase activity were tightly correlated. The biochemical mechanisms of glucose induction of islet glucokinase were further studied. Immunoblotting with an antibody against C-terminal peptide of glucokinase showed that densities of a 52,000-kD protein band from tissue extracts of islets cultured for 7 days in 3, 12, and 30 mM glucose were 25, 44, and 270% compared with that of extract from freshly isolated islets (100%). RNA blot analysis of glucokinase mRNA demonstrated virtually the same levels in fresh islets and islets after 7 days of culture in 3 or 30 mM glucose. The adaptive response of glucokinase to glucose appears therefore to be occurring at a translational or posttranslational site in cultured islets. These data greatly strengthen the concept that glucose is the regulator that induces the activity of glucokinase, which in turn determines the rate change of glucose usage as well as glucose-stimulated insulin release from beta-cells. Thus, the hypothesis that glucokinase is the glucose sensor of beta-cells is strengthened further.     

Tissue-specific deletion of the retinoblastoma protein in the pancreatic beta-cell has limited effects on beta-cell replication, mass, and function

Animal studies show that G(1/S) regulatory molecules (D-cyclins, cdk-4, p18, p21, p27) are critical for normal regulation of beta-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains beta-cell cycle arrest. Logically, removal of pRb from the beta-cell should result in unrestrained beta-cell replication, increased beta-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in beta-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in beta-cells. Surprisingly, although the pRb gene was efficiently recombined in beta-cells of both CKO models, changes in beta-cell mass, beta-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic beta-cell. This may reflect fundamental inaccuracies in models of beta-cell cycle control or complementation for pRb by undefined proteins.