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At least 19 recordsLinked to original sources
PubMed2006

An allosteric activator of glucokinase impairs the interaction of glucokinase and glucokinase regulatory protein and regulates glucose metabolism.

Glucokinase (GK) plays a key role in the control of blood glucose homeostasis. We identified a small molecule GK activator, compound A, that increased the glucose affinity and maximal velocity (V(max)) of GK. Compound A augmented insulin secretion from isolated rat islets and enhanced glucose utilization in primary cultured rat hepatocytes. In rat oral glucose tolerance tests, orally administrated compound A lowered plasma glucose elevation with a concomitant increase in plasma insulin and hepatic glycogen. In liver, GK activity is acutely controlled by its association to the glucokinase regulatory protein (GKRP). In order to decipher the molecular aspects of how GK activator affects the shuttling of GK between nucleus and cytoplasm, the effect of compound A on GK-GKRP interaction was further investigated. Compound A increased the level of cytoplasmic GK in both isolated rat primary hepatocytes and the liver tissues from rats. Experiments in a cell-free system revealed that compound A interacted with glucose-bound free GK, thereby impairing the association of GK and GKRP. On the other hand, compound A did not bind to glucose-unbound GK or GKRP-associated GK. Furthermore, we found that glucose-dependent GK-GKRP interaction also required ATP. Given the combined prominent role of GK on insulin secretion and hepatic glucose metabolism where the GK-GKRP mechanism is involved, activation of GK has a new therapeutic potential in the treatment of type 2 diabetes.

Active Transport, Cell Nucleus↗
PubMed1999

Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase.

Hepatic glucokinase (GK) moves between the nucleus and cytoplasm in response to metabolic alterations. Here, using heterologous cell systems, we have found that at least two different mechanisms are involved in the intracellular movement of GK. In the absence of the GK regulatory protein (GKRP) GK resides only in the cytoplasm. However, in the presence of GKRP, GK moves to the nucleus and resides there in association with this protein until changes in the metabolic milieu prompt its release. GK does not contain a nuclear localization signal sequence and does not enter the nucleus in a GKRP-independent manner because cells treated with leptomycin B, a specific inhibitor of leucine-rich NES-dependent nuclear export, do not accumulate GK in the nucleus. Instead, entry of GK into the nucleus appears to occur via a piggy-back mechanism that involves binding to GKRP. Nuclear export of GK, which occurs after its release from GKRP, is due to a leucine-rich nuclear export signal within the protein ((300)ELVRLVLLKLV(310)). Thus, GKRP appears to function as both a nuclear chaperone and metabolic sensor and is a critical component of a hepatic GK translocation cycle for regulating the activity of this enzyme in response to metabolic alterations.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Anomeric specificity of human liver and B-cell glucokinase: modulation by the glucokinase regulatory protein.

The anomeric specificity of the wild-type recombinant forms of human liver and B-cell glucokinase was investigated using radioactive anomers of d-glucose as tracers. With d-glucose at anomeric equilibrium and at 30 degrees C, the maximal velocity, Hill number, and K(s) amounted, respectively, to 16 micromol min(-1) mg(-1), 1.8 and 6.9 mM in the case of liver glucokinase, and 7.3 micromol min(-1) mg(-1), 2.0 and 7.1 mM in the case of B-cell glucokinase. Whether at 20-22 or 30 degrees C, the maximal velocity, Hill number, and K(m) were significantly lower with alpha-d-glucose than with beta-d-glucose in both liver and B-cell glucokinase. As a result of these differences, the reaction velocity was higher with alpha-d-glucose at low hexose concentrations, while the opposite situation prevailed at high hexose concentrations. In the presence of 0.2 mM d-fructose 6-phosphate, the glucokinase regulatory protein caused a concentration-related inhibition of d-glucose phosphorylation, such an effect fading out at high concentrations of either d-glucose or glucokinase relative to that of its regulatory protein. The phosphorylation of alpha-d-glucose by liver glucokinase appeared more resistant than that of beta-d-glucose to the inhibitory action of d-fructose 6-phosphate, as mediated by the glucokinase regulatory protein. Such a phenomenon failed to achieve statistical significance in the case of the B-cell glucokinase. It is proposed that this information, especially the novel findings concerning the anomeric difference in both Hill number and sensitivity to the glucokinase regulatory protein, should be taken into account when considering the respective contributions of alpha- and beta-d-glucose to the overall phosphorylation of equilibrated d-glucose by glucokinase.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Evidence that glucokinase regulatory protein is expressed and interacts with glucokinase in rat brain.

Our previous description of functional glucokinase isoforms in the rat brain has opened new questions concerning the presence of glucokinase regulatory protein in the brain and the functional role of its interactions with glucokinase. In this study, we found glucokinase regulatory protein mRNA in rat brain, pancreatic islets and liver. In addition, we found two other variant splicing isoforms, both identified in hypothalamus, pancreatic islets and liver. In situ hybridization studies revealed the presence of glucokinase regulatory protein mRNA, the highest number of positive cells being found in the paraventricular nucleus of the hypothalamus. Glucokinase regulatory protein gene expression gave rise to a protein of 69 kDa mainly in nuclear and soluble cell fractions. Glutathione S-transferase protein fused either to rat liver or human pancreatic islet glucokinase were able to precipitate glucokinase regulatory protein from liver or hypothalamic extracts in the presence of fructose-6-phosphate, the amount of protein co-precipitated being decreased with fructose-1-phosphate. These findings suggest that the presence of glucokinase and glucokinase regulatory protein in the rat brain would facilitate the adaptation of this organ to fluctuations in blood glucose concentrations, and both proteins may participate in glucose-sensing and metabolic regulation in the central nervous system.

Adaptor Proteins, Signal Transducing↗
PubMed1987

Molecular cloning of glucokinase cDNA. Developmental and dietary regulation of glucokinase mRNA in rat liver.

A rat liver cDNA library enriched for glucokinase sequences was constructed using the phage expression vector lambda gt11 and screened with an antiserum to glucokinase. A positive phage clone termed lambda-GK223 was isolated by several rounds of plaque purification. When introduced in the high frequency lysogenization strain Y1089, the phage was shown to encode a fusion protein containing epitopes specific to rat liver glucokinase. The 1800-base pair cDNA insert of lambda-GK223 was subcloned in a pUC plasmid, and a resulting recombinant termed pUC-GK1 was used for hybrid selection of mRNA. The selected mRNA directed the synthesis in a cell-free translation system of a protein identified as glucokinase by electrophoresis and immunoprecipitation. The cloned cDNA was then used as a probe to measure the amount of glucokinase mRNA in rat liver during postnatal development. Glucokinase mRNA, 2.4 kilobases in length, was first detectable at day 14 after birth and increased 40-fold in amount from this age to day 31, in parallel with the emergence of glucokinase enzyme activity. In the adult rat, glucokinase mRNA was low during fasting and increased more than 50-fold above the fasting level within 6 h of an oral glucose load. However, maximal accumulation of glucokinase mRNA was short-lived and the mRNA level returned toward basal values by 18 h of refeeding. These data point to rapid and massive effects on the expression of the glucokinase gene at the transcriptional or post-transcriptional levels during ontogenic development and dietary changes in the adult animal.

Animals↗
PubMed2000

A novel glucokinase regulator in pancreatic beta cells: precursor of propionyl-CoA carboxylase beta subunit interacts with glucokinase and augments its activity.

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 beta cells. To search for such a protein in pancreatic beta 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 beta subunit (pbetaPCCase), and glutathione S-transferase pull-down assay illustrated that pbetaPCCase interacted with recombinant rat islet glucokinase and with glucokinase in rat liver and islet extracts. Functional analysis indicated that pbetaPCCase decreased the K(m) value of recombinant islet glucokinase for glucose by 18% and increased V(max) value by 23%. We concluded that pbetaPCCase might be a novel activator of glucokinase in pancreatic beta cells.

Animals↗
PubMed1996

Cloning and biochemical characterisation of an Aspergillus niger glucokinase. Evidence for the presence of separate glucokinase and hexokinase enzymes.

The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a pI of 5.2. The enzyme has high affinity for glucose (K(m) 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K(m) 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The kcal (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate.

Adenosine Diphosphate↗
PubMed2006

The network of glucokinase-expressing cells in glucose homeostasis and the potential of glucokinase activators for diabetes therapy.

The glucose-phosphorylating enzyme glucokinase has structural, kinetic, and molecular genetic features that are ideal for its primary role as glucose sensor in a network of neuro/endocrine sentinel cells that maintain glucose homeostasis in many vertebrates including humans. The glucokinase-containing, insulin-producing beta-cells of the pancreas take the prominent lead in this network, functioning in the aggregate as the master gland. The beta-cells are also conceptualized as the prototype for all other glucose sensor cells, which determines our current understanding of many extrapancreatic glucose sensors. About 99% of the enzyme resides, however, in the hepato-parenchymal cells and serves its second role in a high-capacity process of blood glucose clearance. Two examples strikingly illustrate how pivotal a position glucokinase has in the regulation of glucose metabolism: 1) activating and inactivating mutations of the enzyme cause hypo- and hyperglycemia syndromes in humans described collectively as "glucokinase disease" and fully explained by the glucose sensor paradigm, and 2) glucokinase activator drugs (GKAs) have been discovered that bind to an allosteric site and increase the kcat and lower the glucose S(0.5) of the enzyme. GKAs enhance glucose-stimulated insulin release from pancreatic islets and glucose disposition by the liver. They are now intensively explored to develop a novel treatment for diabetes. Future biophysical, molecular, genetic, and pharmacological studies hold much promise to unravel the evolving complexity of the glucokinase glucose sensor system.

Diabetes Mellitus↗
PubMed1999

Inhibitory effects of streptozotocin, tumor necrosis factor-alpha, and interleukin-1beta on glucokinase activity in pancreatic islets and gene expression of GLUT2 and glucokinase.

Treatment of streptozotocin (ST), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) resulted in destroying insulin-secreting beta-cells of pancreatic islets and impairment of islet glucose oxidation and glucose-induced insulin secretion. IL-1beta and TNF-alpha inhibited insulin release and glucose utilization and oxidation. It was shown that the inhibitory effects of ST, IL-1beta, and TNF-alpha were due to impaired glucokinase activity. Glucokinase activity was severely impaired by ST, IL-1beta, and TNF-alpha treatments, as confirmed by assaying enzymes and nucleotides associated with glycolysis and glucose oxidation. On the other hand, nitric oxide was a factor of the deleterious effects of IL-1beta, TNF-alpha, and ST on pancreatic islets. Incubation of mouse pancreatic islets with ST at various concentrations of impairing insulin secretion resulted in generation of nitrite, stimulation of islet guanylyl cyclase and accumulation of cGMP, and inhibition of pancreatic islet mitochondrial aconitase activity to degree similar to those raised by IL-1beta and TNF-alpha. When the effects of IL-1beta and TNF-alpha on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. This suggested that IL-1beta and TNF-alpha downregulate gene expression of GLUT2 and glucokinase in pancreatic beta-cells.

Aconitate Hydratase↗
PubMed1976

Role of ATP-glucokinase and polyphosphate glucokinase in Streptomyces aureofaciens.

The activity of ATP-glucokinase and of polyphosphate glucokinase was examined during growth of the actinomycete Streptomyces aureofaciens 8425 under conditions of intense chlortetracycline (CTC) synthesis. ATP-glucokinase was active in the strain only during the logarithmic phase of culture growth; the activity of polyphosphate glucokinase appears only at the end of the logarithmic phase of growth and rises in parallel with the rate of CTC biosynthesis in the stationary phase. During the rise of activity of polyphosphate glucokinase and of CTC biosynthesis the cells accumulate sugar phosphates, mainly glucose-6-phosphate. It appears that the biosynthesis of CTC in Streptomyces aureofaciens takes place at the expense of glycolysis, using up the high-energy phosphate of high-molecular polyphosphates.

Adenosine Triphosphate↗
PubMed2000

Glucokinase and glucokinase regulatory protein: mutual dependence for nuclear localization.

Conditional expression of the glucokinase regulatory protein in insulinoma cells, under control of the reverse tetracycline-dependent transactivator, was used to investigate whether expression of this protein de novo would alter the intracellular distribution of glucokinase. The regulatory protein, which was undetectable in the basal state, could be induced by doxycycline to levels comparable to those of liver and was detected mostly in the nucleus. Concomitantly, glucokinase accumulated in the nucleus. Human embryonic kidney cells were transiently transfected to express glucokinase and the regulatory protein, either separately or together. Each protein localized predominantly to the cytoplasm when expressed alone. On co-expression, however, both proteins localized virtually entirely to the nucleus. The enzymic activity of glucokinase was not required for promoting nuclear import of the two proteins, as shown with a glucose-phosphorylation-deficient mutant. Finally, in embryonic kidney cells expressing the regulatory protein alone, treatment with leptomycin B resulted in a partial redistribution of the protein from the cytoplasm to the nucleus, suggesting that this protein can shuttle between the two compartments.

Adaptor Proteins, Signal Transducing↗
PubMed1995

The regulatory protein of glucokinase binds to the hepatocyte matrix, but, unlike glucokinase, does not translocate during substrate stimulation.

The kinetic properties of hepatic glucokinase (hexokinase IV) are modulated by binding to a regulatory protein. This study shows that, in hepatocytes incubated with 5 mM glucose as sole carbohydrate substrate, both glucokinase and its regulatory protein bind to the cell matrix by a Mg(2+)-dependent mechanism. After incubation with an elevated [glucose] or with fructose, glucokinase, but not its regulatory protein, translocates from the Mg(2+)-dependent binding site. It is suggested that the regulatory protein acts as a receptor for anchoring glucokinase to the hepatocyte matrix and inhibiting its activity in metabolically quiescent conditions.

Animals↗
PubMed2002

The first archaeal ATP-dependent glucokinase, from the hyperthermophilic crenarchaeon Aeropyrum pernix, represents a monomeric, extremely thermophilic ROK glucokinase with broad hexose specificity.

An ATP-dependent glucokinase of the hyperthermophilic aerobic crenarchaeon Aeropyrum pernix was purified 230-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of about 36 kDa. The apparent K(m) values for ATP and glucose (at 90 degrees C and pH 6.2) were 0.42 and 0.044 mM, respectively; the apparent V(max) was about 35 U/mg. The enzyme was specific for ATP as a phosphoryl donor, but showed a broad spectrum for phosphoryl acceptors: in addition to glucose, which showed the highest catalytic efficiency (k(cat)/K(m)), the enzyme also phosphorylates glucosamin, fructose, mannose, and 2-deoxyglucose. Divalent cations were required for maximal activity: Mg(2+), which was most effective, could partially be replaced with Co(2+), Mn(2+), and Ni(2+). The enzyme had a temperature optimum of at least 100 degrees C and showed significant thermostability up to 100 degrees C. The coding function of open reading frame (ORF) APE2091 (Y. Kawarabayasi, Y. Hino, H. Horikawa, S. Yamazaki, Y. Haikawa, K. Jin-no, M. Takahashi, M. Sekine, S. Baba, A. Ankai, H. Kosugi, A. Hosoyama, S. Fukui, Y. Nagai, K. Nishijima, H. Nakazawa, M. Takamiya, S. Masuda, T. Funahashi, T. Tanaka, Y. Kudoh, J. Yamazaki, N. Kushida, A. Oguchi, and H. Kikuchi, DNA Res. 6:83-101, 145-152, 1999), previously annotated as gene glk, coding for ATP-glucokinase of A. pernix, was proved by functional expression in Escherichia coli. The purified recombinant ATP-dependent glucokinase showed a 5-kDa higher molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but almost identical kinetic and thermostability properties in comparison to the native enzyme purified from A. pernix. N-terminal amino acid sequence of the native enzyme revealed that the translation start codon is a GTG 171 bp downstream of the annotated start codon of ORF APE2091. The amino acid sequence deduced from the truncated ORF APE2091 revealed sequence similarity to members of the ROK family, which comprise bacterial sugar kinases and transcriptional repressors. This is the first report of the characterization of an ATP-dependent glucokinase from the domain of Archaea, which differs from its bacterial counterparts by its monomeric structure and its broad specificity for hexoses.

Adenosine Triphosphate↗
PubMed2000

Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose.

To identify the amino acids involved in the specific regulatory properties of glucokinase, and particularly its low affinity for glucose, mutants of the human islet enzyme have been prepared, in which glucokinase-specific residues have been replaced. Two mutations increased the affinity for glucose by twofold (K296M) and sixfold (Y214A), the latter also decreasing the Hill coefficient from 1.75 to 1.2 with minimal change in the affinity for ATP. Combining these two mutations with N166R resulted in a 50-fold decrease in the half-saturating substrate concentration (S0.5) value, which became then comparable to the Km of hexokinase II. The location of N166, Y214, and K296 in the three-dimensional structure of glucokinase suggests that these mutations act by favoring closure of the catalytic cleft. As a rule, mutations changed the affinity for glucose and for the competitive inhibitor mannoheptulose (MH) in parallel, whereas they barely affected the affinity for N-acetylglucosamine (NAG). These and other results suggest that NAG and MH bind to the same site but to different conformations of glucokinase. A small reduction in the affinity for the regulatory protein was observed with mutations of residues on the smaller domain and in the hinge region, confirming the bipartite nature of the binding site for the regulatory protein. The K296M mutant was found to have a threefold decreased affinity for palmitoyl CoA; this effect was additive to that previously observed for the E279Q mutant, indicating that the binding site for long-chain acyl CoAs is located on the upper face of the larger domain.

Adenosine Triphosphate↗
PubMed2006

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity.

AIMS/HYPOTHESIS: Glucokinase (GCK) acts as a glucose sensor in the pancreatic beta cell and regulates insulin secretion. In the gene encoding GCK the heterozygous mutations that result in enzyme inactivation cause MODY2. Functional studies of naturally occurring GCK mutations associated with hyperglycaemia provide further insight into the biochemical basis of glucose sensor regulation. MATERIALS AND METHODS: Identification of GCK mutations in selected MODY patients was performed by single-strand conformation polymorphism and direct sequencing. The kinetic parameters and thermal stability of recombinant mutant human GCK were determined, and in pull-down assays the effect of these mutations on the association of GCK with glucokinase (hexokinase 4) regulator (GCKR, also known as glucokinase regulatory protein [GKRP]) and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB1, also known as PFK2) was tested. RESULTS: We identified three novel GCK mutations: the insertion of an asparagine residue at position 161 (inserN161) and two missense mutations (M235V and R308W). We also identified a fourth mutation (R397L) reported in a previous work. Functional characterisation of these mutations revealed that insertion of asparagine residue N161 fully inactivates GCK, whereas the M235V and R308W mutations only partially impair enzymatic activity. In contrast, GCK kinetics was almost unaffected by the R397L mutation. Although none of these mutations affected the interaction of GCK with PFKFB1, we found that the R308W mutation caused protein instability and increased the strength of interaction with GCKR. CONCLUSIONS/INTERPRETATION: Our results show that different MODY2 mutations impair GCK function through different mechanisms such as enzymatic activity, protein stability and increased interaction with GCKR, helping further elucidate the regulation of GCK activity.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Intracellular distribution of hepatic glucokinase and glucokinase regulatory protein during the fasted to refed transition in rats.

We have studied the intracellular distribution in vivo of glucokinase (GK) and glucokinase regulatory protein (GKRP) in livers of fasted and refed rats, using specific antibodies against both proteins and laser confocal fluorescence microscopy. GK was found predominantly in the nucleus of hepatocytes from starved rats. GK was translocated to the cytoplasm in livers of 1- and 2-h refed animals, but returned to the nucleus after 4 h. GKRP concentrated in the hepatocyte nuclei and its distribution did not change upon refeeding. These results show that, in physiological conditions, GKRP is present predominantly in the nuclei of hepatocytes and that the translocation of hepatic GK from and to the nucleus is operative in vivo.

Animals↗
PubMed2006

Biochemical basis of glucokinase activation and the regulation by glucokinase regulatory protein in naturally occurring mutations.

Glucokinase (GK) has several known polymorphic activating mutations that increase the enzyme activity by enhancing glucose binding affinity and/or by alleviating the inhibition of glucokinase regulatory protein (GKRP), a key regulator of GK activity in the liver. Kinetic studies were undertaken to better understand the effect of these mutations on the enzyme mechanism of GK activation and GKRP regulation and to relate the enzyme properties to the associated clinical phenotype of hypoglycemia. Similar to wild type GK, the transient kinetics of glucose binding for activating mutations follows a general two-step mechanism, the formation of an enzyme-glucose complex followed by an enzyme conformational change. However, the kinetics for each step differed from wild type GK and could be grouped into specific types of kinetic changes. Mutations T65I, Y214C, and A456V accelerate glucose binding to the apoenzyme form, whereas W99R, Y214C, and V455M facilitate enzyme isomerization to the active form. Mutations that significantly enhance the glucose binding to the apoenzyme also disrupt the protein-protein interaction with GKRP to a large extent, suggesting these mutations may adopt a more compact conformation in the apoenzyme favorable for glucose binding. Y214C is the most active mutation (11-fold increase in k(cat)/K(0.5)(h)) and exhibits the most severe clinical effects of hypoglycemia. In contrast, moderate activating mutation A456V nearly abolishes the GKRP inhibition (76-fold increase in K(i)) but causes only mild hypoglycemia. This suggests that the alteration in GK enzyme activity may have a more profound biological impact than the alleviation of GKRP inhibition.

Allosteric Site↗
PubMed1997

Glucokinase regulatory protein may interact with glucokinase in the hepatocyte nucleus.

Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic beta-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

1-Methyl-3-isobutylxanthine↗