An Allosteric Activator of Glucokinase Impairs the Interaction of Glucokinase and Glucokinase Regulatory Protein and Regulates Glucose Metabolism

Abstract

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 GKRPassociated 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.There are three key aspects of type 2 diabetes pathogenesis, which are the focus of current and future therapies: insulin resistance, defective insulin secretion, and increased hepatic glucose production. Glucokinase (GK) 2 is the predominant glucose phosphorylation enzyme in pancreatic ␤-cells and hepatocytes. GK plays an important role as a glucose sensor for controlling plasma glucose homeostasis by enhancing insulin secretion from pancreatic ␤-cells and glucose metabolism in the liver (1, 2), which provides rational expectations that enhancement of GK activity would be a novel therapeutic strategy for type 2 diabetes. Consistent with this rationale, recently discovered small molecule allosteric activators of GK have been demonstrated to have antidiabetic efficacy in rodents (3, 4).To investigate the mechanism of action of GK activators, the interaction between GK and glucokinase regulatory protein (GKRP) is a key aspect. It is well known that hepatic GK activity is modulated by the endogenous inhibitor, glucokinase regulatory protein (GKRP) (5-8). GK is localized in the nucleus as an inactive complex with GKRP at low glucose concentrations and is dissociated from the GK⅐GKRP complex and translocated to the cytoplasm at high glucose concentrations, which triggers glucose disposal (9). Modulators of the GK-GKRP interaction have been shown to enhance hepatic glucose disposal (7). We recently solved the co-crystal structure of hepatic GK complex with GK activator, in which GK undergoes a large conformational change between the active and inactive forms at diffe...

Molecular basis for the role of glucokinase regulatory protein as the allosteric switch for glucokinase

“…S6). This result indicates that GKA dissociates GKRP (7,9,17,23) through a structural rearrangement of GK.…”

“…It is noteworthy that F1P is likely to be more efficient than glucose or GKA in releasing GK from GKRP. Although F1P, glucose, and GKA bind to distant sites from the interface of the GK/GKRP complex (9), F1P induces direct disruption of the interface to release GK from GKRP, whereas glucose and GKA induce slow and energetically unfavorable rearrangement of the GK small domain (9,17). The superimposition of F6P-bound hGKRP onto apohGKRP revealed that the interaction between the hydroxyl group of C1 in F6P and Glu348 of α10 in SIS domain 2 shifts α10 downward (Fig.…”

“…F1P is derived from fructose and sorbitol in a meal, and F6P is an intermediate product of glycolysis (16). Recent studies have characterized the biophysical features regarding the interaction between GK and GKRP and a modulation through the effectors (17,18). Despite the intensive studies on GK and GKRP, the molecular basis for the allosteric regulation mechanism of GK by GKRP remains poorly understood because of the lack of structural information of the GK/GKRP complex.…”

Control of beta cell function and proliferation in mice stimulated by small-molecule glucokinase activator under various conditions

“…Since the report by Grimsby et al in 2003 [5], several glucokinase activators (GKAs) have been developed, and these have been shown to lower blood glucose in several animal models of type 2 diabetes [5][6][7][8][9][10][11][12]. In a study of beta cell function, it was reported that a GKA stimulated insulin secretion in a Ca 2+ -dependent manner in rodent islets and MIN6 cells [13], and we and others have reported that GKAs promoted beta cell proliferation and increased production of IRS2 [11,14], which is critically required for beta cell growth and survival [3,[15][16][17].…”

Chronic treatment with a glucokinase activator delays the onset of hyperglycaemia and preserves beta cell mass in the Zucker diabetic fatty rat

“…GKAs have demonstrated prominent antihyperglycaemic activity in animal models [12][13][14][15] and in clinical trials [16,17]. It is, however, currently uncertain whether long-term improvements in glycaemic control can be achieved with these agents.…”