Mutational analysis of allosteric activation and inhibition of glucokinase.

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.

Heme-oxygenase induction inhibits arteriolar thrombosis in vivo: effect of the non-substrate inducer cobalt protoporphyrin.

Heme oxygenase-1 (HO) metabolizes heme to form the vasodilator carbon monoxide and antioxidant biliverdin. Upregulation of HO-1 by hemin, which is also a substrate attenuates thrombosis in rodent models, however, whether protection is due to HO-1 upregulation or to increased substrate availability is unknown. This study tested the hypothesis that treatment of mice with cobalt protoporphyrin (CoPP), a non-substrate HO-1 inducer, would protect the endothelium from laser injury. C57Bl/J6 mice were treated with vehicle, CoPP (20 mg/kg), CoPP plus the HO-1 inhibitor tin protoporphyrin (SnPP; 20 mg/kg) or SnPP alone for 18 h. Intravital microscopy was used to quantitate thrombus formation in cremaster arterioles in response to laser ablation of the endothelium. CoPP treatment inhibited thrombosis by 43% compared to vehicle (P<0.05). SnPP co-treatment negated the inhibitory effect of CoPP while SnPP alone potentiated thrombosis compared to vehicle. In CoPP-treated animals, cremaster HO-1 mRNA expression was increased 59+/-17-fold over vehicle (P<0.001). Co-treatment with CoPP+SnPP attenuated this effect by 36%, however the increase in HO-1 protein induced by CoPP was unaffected by SnPP. Induction of HO-1 by the non-substrate inducer CoPP protects against laser induced endothelial injury without the need for increased substrate. Small molecule, substrate-independent upregulation of HO-1 expression represents a feasible approach to ameliorate endothelial dysfunction in cardiovascular disease.

A glucose sensor role for glucokinase in anterior pituitary cells.

Enzymatic activity of glucokinase was demonstrated, quantitated, and characterized kinetically in rat and mouse pituitary extracts using a highly specific and sensitive spectrometric assay. A previously proposed hypothesis that the glucokinase gene might be expressed in the pituitary corticotrophic cells was therefore reexamined using mRNA in situ hybridization and immunohistochemical techniques. No evidence was found that corticotrophs are glucokinase positive, and the identity of glucokinase-expressing cells remains to be determined. The findings do, however, suggest a novel hypothesis that a critical subgroup of anterior pituitary cells might function as glucose sensor cells and that direct fuel regulation of such cells may modify the classical indirect neuroendocrine pathways that are known to control hormone secretion from anterior pituitary cells.

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.

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.

Insights into the structure and regulation of glucokinase from a novel mutation (V62M), which causes maturity-onset diabetes of the young.

Glucokinase (GCK) serves as the pancreatic glucose sensor. Heterozygous inactivating GCK mutations cause hyperglycemia, whereas activating mutations cause hypoglycemia. We studied the GCK V62M mutation identified in two families and co-segregating with hyperglycemia to understand how this mutation resulted in reduced function. Structural modeling locates the mutation close to five naturally occurring activating mutations in the allosteric activator site of the enzyme. Recombinant glutathionyl S-transferase-V62M GCK is paradoxically activated rather than inactivated due to a decreased S0.5 for glucose compared with wild type (4.88 versus 7.55 mM). The recently described pharmacological activator (RO0281675) interacts with GCK at this site. V62M GCK does not respond to RO0281675, nor does it respond to the hepatic glucokinase regulatory protein (GKRP). The enzyme is also thermally unstable, but this lability is apparently less pronounced than in the proven instability mutant E300K. Functional and structural analysis of seven amino acid substitutions at residue Val62 has identified a non-linear relationship between activation by the pharmacological activator and the van der Waals interactions energies. Smaller energies allow a hydrophobic interaction between the activator and glucokinase, whereas larger energies prohibit the ligand from fitting into the binding pocket. We conclude that V62M may cause hyperglycemia by a complex defect of GCK regulation involving instability in combination with loss of control by a putative endogenous activator and/or GKRP. This study illustrates that mutations that cause hyperglycemia are not necessarily kinetically inactivating but may exert their effects by other complex mechanisms. Elucidating such mechanisms leads to a deeper understanding of the GCK glucose sensor and the biochemistry of beta-cells and hepatocytes.

Restitution of defective glucose-stimulated insulin release of sulfonylurea type 1 receptor knockout mice by acetylcholine.

Inhibition of ATP-sensitive K+ (K(ATP)) channels by an increase in the ATP/ADP ratio and the resultant membrane depolarization are considered essential in the process leading to insulin release (IR) from pancreatic beta-cells stimulated by glucose. It is therefore surprising that mice lacking the sulfonylurea type 1 receptor (SUR1-/-) in beta-cells remain euglycemic even though the knockout is expected to cause hypoglycemia. To complicate matters, isolated islets of SUR1-/- mice secrete little insulin in response to high glucose, which extrapolates to hyperglycemia in the intact animal. It remains thus unexplained how euglycemia is maintained. In recognition of the essential role of neural and endocrine regulation of IR, we evaluated the effects of acetylcholine (ACh) and glucagon-like peptide-1 (GLP-1) on IR and free intracellular Ca2+ concentration ([Ca2+]i) of freshly isolated or cultured islets of SUR1-/- mice and B6D2F1 controls (SUR1+/+). IBMX, a phosphodiesterase inhibitor, was also used to explore cAMP-dependent signaling in IR. Most striking, and in contrast to controls, SUR1-/-) islets are hypersensitive to ACh and IBMX, as demonstrated by a marked increase of IR even in the absence of glucose. The hypersensitivity to ACh was reproduced in control islets by depolarization with the SUR1 inhibitor glyburide. Pretreatment of perifused SUR1-/- islets with ACh or IBMX restored glucose stimulation of IR, an effect expectedly insensitive to diazoxide. The calcium channel blocker verapamil reduced but did not abolish ACh-stimulated IR, supporting a role for intracellular Ca2+ stores in stimulus-secretion coupling. The effect of ACh on IR was greatly potentiated by GLP-1 (10 nM). ACh caused a dose-dependent increase in [Ca2+]i at 0.1-1 microM or biphasic changes (an initial sharp increase in [Ca2+]i followed by a sustained phase of low [Ca2+]i) at 1-100 microM. The latter effects were observed in substrate-free medium or in the presence of 16.7 mM glucose. We conclude that SUR1 deletion depolarizes the beta-cells and markedly elevates basal [Ca2+]i. Elevated [Ca2+]i in turn sensitizes the beta-cells to the secretory effects of ACh and IBMX. Priming by the combination of high [Ca2+]i, ACh, and GLP-1 restores the defective glucose responsiveness, precluding the development of diabetes but not effectively enough to cause hyperinsulinemic hypoglycemia.