Two birds with one stone: novel glucokinase activator stimulates glucose-induced pancreatic insulin secretion and augments hepatic glucose metabolism

The hormones glucagon and insulin delicately regulate the concentration of blood glucose. When patients become resistant to the effects of insulin or produce too little of it to properly regulate glucose concentrations, then diabetes can result. Unfortunately, not all patients with insulin-resistant, type 2 diabetes mellitus respond to drugs that improve insulin sensitivity. However, there is reason to be hopeful. A new molecule that targets glucokinase (GK), the enzyme responsible for phosphorylating glucose in pancreatic beta cells and hepatic cells, acts to significantly reduce blood glucose concentrations in rodents. The GK activator RO-28-1675 increased the glucose affinity and Vmax of GK, and rats treated with RO-28-1675 had improved glucose tolerance and elevated glucose uptake in liver. These results provide the basis for improved drug design that may alleviate diabetes mellitus and the disorders that accompany it in patients.     

Regulation of Endogenous Glucose Production in Glucose Transporter 4 Over-Expressing Mice

Strategies to amplify whole-body glucose disposal are key therapies to treat type 2 diabetes. Mice that over-express glucose transporter 4 (Glut4) in skeletal muscle, heart, and adipose tissue (G4Tg) exhibit increased fasting glucose disposal and thus lowered blood glucose. Intriguingly, G4Tg mice also exhibit improved insulin-stimulated suppression of endogenous glucose production even though Glut4 is not present in the liver. It is unclear, however, if hepatic gluco-regulation is altered in G4Tg mice in the basal, non-insulin-stimulated state. The current studies were performed to examine fasting hepatic glucose metabolism in G4Tg mice and to determine whether gluco-regulatory adaptations exist in the non-insulin-stimulated condition. To test this question, phloridzin-glucose clamps were used to match blood glucose and pancreatic hormone levels while tracer dilution techniques were used to measure glucose flux. These techniques were performed in chronically-catheterized, conscious, and un-stressed 5h-fasted G4Tg and wild-type (WT) littermates. Results show reduced blood glucose, hepatic glycogen content, and hepatic glucokinase (GK) activity/expression as well as higher endogenous glucose production, glucose disposal, arterial glucagon, and hepatic glucose-6-phosphatase (G6Pase) activity/expression in G4Tg mice versus WT controls. Clamping blood glucose for 90 min at ∼115 mg/dLin G4Tg and WT mice normalized nearly all variables. Notably, however, net hepatic glycogen synthetic rates were disproportionately elevated compared to changes in blood glucose. In conclusion, these studies demonstrate that basal improvements in glucose tolerance due to increased uptake in extra-hepatic sites provoke important gluco-regulatory adaptations in the liver. Although changes in blood glucose underlie the majority of these adaptations, net hepatic glycogen synthesis is sensitized. These data emphasize that anti-diabetic therapies that target skeletal muscle, heart, and/or adipose tissue likely positively impact the liver.     

Changes in cephalic and peripheral use of glucose and glutamine under the influence of hyperammonemia in rats]

Arterio-venous differences of glucose and glutamine were determined across the brain and across the hind limb in normal and ammonium salt infused rats, before and during an insulin tolerance test, in an attempt to study the effect of hyperammonemia on cephalic and muscular metabolism. The results demonstrate that 1) hyperammonemia reduces the hind limb uptake of glucose without affect the cephalic uptake of glucose which is lowered during hypoglycemia, 2) the reduction of the cephalic and muscular glutamine output induced by the hypoglycemia is masked in presence of an hyperammonemia. In conclusion, it may be assume that, at the concentration obtained in this study, hyperammonemia does not act directly in the pathogenesis of hepatic coma in which a decrease in cerebral glucose uptake described; on the other hand, ammonium plays an important role in the muscle metabolism.     

Alpha-lipoic acid mitigates insulin resistance in Goto-Kakizaki rats.

Impaired glucose uptake and metabolism by peripheral tissues is a common feature in both type I and type II diabetes mellitus. This phenomenon was examined in the context of oxidative stress and the early events within the insulin signalling pathway using soleus muscles derived from non-obese, insulin-resistant type II diabetic Goto-Kakizaki (GK) rats, a well-known genetic rat model for human type II diabetes. Insulin-stimulated glucose transport was impaired in soleus muscle from GK rats. Oxidative and non-oxidative glucose disposal pathways represented by glucose oxidation and glycogen synthesis in soleus muscles of GK rats appear to be resistant to the action of insulin when compared to their corresponding control values. These diabetes-related abnormalities in glucose disposal were associated with a marked diminution in the insulin-mediated enhancement of protein kinase B (Akt/PKB) and insulin receptor substrate-1 (IRS-1)-associated phosphatidylinostol 3-kinase (PI 3-kinase) activities; these two kinases are key elements in the insulin signalling pathway. Moreover, heightened state of oxidative stress, as indicated by protein bound carbonyl content, was evident in soleus muscle of GK diabetic rats. Chronic administration of the hydrophobic/hydrophilic antioxidant alpha -lipoic-acid (ALA, 100 mg/kg, i.p.) partly ameliorated the diabetes-related deficit in glucose metabolism, protein oxidation as well as the activation by insulin of the various steps of the insulin signalling pathway, including the enzymes Akt/PKB and PI-3 kinase. Overall, the current investigation illuminates the concept that oxidative stress may indeed be involved in the pathogenesis of certain types of insulin resistance. It also harmonizes with the notion of including potent antioxidants such as ALA in the armamentarium of antidiabetic therapy.     

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.     

GLUT4: a key player regulating glucose homeostasis? Insights from transgenic and knockout mice (review).

Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in skeletal muscle is important to develop effective strategies to prevent or reduce the incidence of Type II diabetes mellitus. In patients with Type II diabetes mellitus, reduced glucose transport in skeletal muscle is a major factor responsible for reduced whole body glucose uptake. Overexpression of GLUT4 in skeletal muscle improves glucose homeostasis in animal models of diabetes mellitus and protects against the development of diabetes mellitus. Thus, GLUT4 is an attractive target for pharmacological intervention strategies to control glucose homeostasis. This review will focus on the current understanding of the role of GLUT4 in regulating cellular glucose uptake and whole body glucose homeostasis.     

Endothelial Na+-D-glucose cotransporter: no role in insulin-mediated glucose uptake.

A recent report indicates that the Na+-D-glucose cotransporter SGLT1 is present in capillaries of skeletal muscle and is required for insulin-mediated glucose uptake in myocytes. This result is based on the complete inhibition of insulin-mediated muscle glucose uptake by phlorizin, an inhibitor of SGLT1. Using the pump-perfused rat hind limb, we measured glucose uptake, lactate efflux, and radioactive 2-deoxyglucose uptake into individual muscles with saline (control), phlorizin, insulin, and insulin plus phlorizin, as well as with saline and insulin using normal and low Na+ perfusion buffer. Insulin-mediated glucose uptake was not inhibited after correction for phlorizin interference in the glucose assay. Lactate efflux and 2-deoxyglucose uptake by individual muscles were unaffected by phlorizin. Low Na+ buffer did not affect insulin-mediated glucose uptake, lactate efflux, or 2-deoxyglucose uptake. We conclude that endothelial SGLT1 exerts no barrier for glucose delivery to myocytes.     

GLUT4: a key player regulating glucose homeostasis? Insights from transgenic and knockout mice

Studies in which GLUT4 has been overexpressed in transgenic mice provide definitive evidence that glucose transport is rate limiting for muscle glucose disposal. Transgenic overexpression of GLUT4 selectively in skeletal muscle results in increased whole body glucose uptake and improves glucose homeostasis. These studies strengthen the hypothesis that the level of muscle GLUT4 affects the rate of whole body glucose disposal, and underscore the importance of GLUT4 in skeletal muscle for maintaining whole body glucose homeostasis. Studies in which GLUT4 has been ablated or 'knocked-out' provide proof that GLUT4 is the primary effector for mediating glucose transport in skeletal muscle and adipose tissue. Genetic ablation of GLUT4 results in impaired insulin tolerance and defects in glucose metabolism in skeletal muscle and adipose tissue. Because impaired muscle glucose transport leads to reduced whole body glucose uptake and hyperglycaemia, understanding the molecular regulation of glucose transport in skeletal muscle is important to develop effective strategies to prevent or reduce the incidence of Type II diabetes mellitus. In patients with Type II diabetes mellitus, reduced glucose transport in skeletal muscle is a major factor responsible for reduced whole body glucose uptake. Overexpression of GLUT4 in skeletal muscle improves glucose homeostasis in animal models of diabetes mellitus and protects against the development of diabetes mellitus. Thus, GLUT4 is an attractive target for pharmacological intervention strategies to control glucose homeostasis. This review will focus on the current understanding of the role of GLUT4 in regulating cellular glucose uptake and whole body glucose homeostasis.     

Effect of early dietary restriction on insulin action and secretion in the GK rat, a spontaneous model of NIDDM

The availability of the Goto-Kakisaki (GK) rat model of non-insulin-dependent diabetes mellitus prompted us to test the effect of a limited period of undernutrition in previously diabetic young rats on their insulin secretion and insulin action during adult age. Four-week-old female GK rats were either food restricted (35% restriction, 15% protein diet) or protein and energy restricted (35% restriction, 5% protein diet) for 4 wk. Food restriction in the young GK rat lowered weight gain but did not aggravate basal hyperglycemia or glucose intolerance, despite a decrease in basal plasma insulin level. Furthermore, the insulin-mediated glucose uptake by peripheral tissues in the GK rat was clearly improved. We also found that food restriction, when it is coupled to overt protein deficiency in the young GK rat, altered weight gain more severely and slightly decreased basal hyperglycemia but conversely aggravated glucose tolerance. Improvement of basal hyperglycemia was related to repression of basal hepatic glucose hyperproduction, despite profound attenuation of basal plasma insulin level. Deterioration of tolerance to glucose was related to severe blunting of the residual glucose-induced insulin secretion. It is, however, likely that the important enhancement of the insulin-mediated glucose uptake helped to limit the deterioration of glucose tolerance.     

Identification of an intracellular pool of glucose transporters from basal and insulin-stimulated rat skeletal muscle

The purpose of this study was to simultaneously isolate skeletal muscle plasma and microsomal membranes from the hind limbs of male Sprague-Dawley rats perfused either in the absence or presence of 20 milliunits/ml insulin and to determine the effect of insulin on the number and distribution of glucose transporters in these membrane fractions. Insulin increased hind limb glucose uptake greater than 3-fold (2.4 +/- 0.7 versus 9.2 +/- 1.0 mumol/g x h, p less than 0.001). Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased 2-fold (9.1 +/- 1.0 to 20.4 +/- 3.1 pmol/mg protein, p less than 0.005) in insulin-stimulated muscle while microsomal membrane transporters decreased significantly (14.8 +/- 1.6 to 9.8 +/- 1.4 pmol/mg protein, p less than 0.05). No change in the dissociation constant (Kd approximately 120 nm) was observed. K+-stimulated-p-nitrophenol phosphatase, 5'-nucleotidase, and galactosyltransferase specific activity, enrichment, and recovery in the plasma and microsomal membrane fractions were not altered by insulin treatment. Western blot analysis using the monoclonal antibody mAb 1F8 (specific for the insulin-regulatable glucose transporter) demonstrated increased glucose transporter densities in plasma membranes from insulin-treated hind limb skeletal muscle compared with untreated tissues, while microsomal membranes from the insulin-treated hind limb skeletal muscle had a concomitant decrease in transporter density. We conclude that the increase in plasma membrane glucose transporters explains, at least in part, the increase in glucose uptake associated with insulin stimulation of hind limb skeletal muscle. Our data further suggest that these recruited transporters originate from an intracellular microsomal pool, consistent with the translocation hypothesis.     

Respiratory chain dysfunction in skeletal muscle does not cause insulin resistance

Insulin resistance in skeletal muscle is a characteristic feature of diabetes mellitus type 2 (DM2). Several lines of circumstantial evidence suggest that reduced mitochondrial oxidative phosphorylation capacity in skeletal muscle is a primary defect causing insulin resistance and subsequent development of DM2. We have now experimentally tested this hypothesis by characterizing glucose homeostasis in tissue-specific knockout mice with progressive respiratory chain dysfunction selectively in skeletal muscle. Surprisingly, these knockout mice are not diabetic and have an increased peripheral glucose disposal when subjected to a glucose tolerance test. Studies of isolated skeletal muscle from knockout animals show an increased basal glucose uptake and a normal increase of glucose uptake in response to insulin. In summary, our findings indicate that mitochondrial dysfunction in skeletal muscle is not a primary etiological event in DM2.     

Oxidative stress--mediated alterations in glucose dynamics in a genetic animal model of type II diabetes

Insulin resistance, characterized by an inexorable decline in skeletal muscle glucose utilization and/or an excessive hepatic glucose production, constitutes a major pathogenic importance in a cluster of clinical disorders including diabetes mellitus, hypertension, dyslipidemia, central obesity and coronary artery disease. A novel concept suggests that heightened state of oxidative stress during diabetes contributes, at least in part, to the development of insulin resistance. Several key predictions of this premise were subjected to experimental testing using Goto-Kakizaki (GK) rats as a genetic animal model for non-obese type II diabetes. Euglycemic-hyperinsulinemic clamp studies with an insulin infusion index of 5 mU/kg bw/min were used to measure endogenous glucose production (EGP), glucose infusion rate (GIR), glucose disposal rate (GDR) and skeletal muscle glucose utilization index (GUI). Moreover, the status of oxidative stress as reflected by the urinary levels of isoprostane and protein carbonyl formation were also assessed as a function of diabetes. Post-absorptive basal EGP and circulating levels of insulin, glucose and free fatty acid (FFA) were elevated in GK rats, compared to their corresponding control values. In contrast, steady state GIR and GDR of the hyperglycemic/hyperinsulinemic animals were reduced, concomitantly with impaired insulin's ability to suppress EGP. Insulin stimulated [3H]-2-deoxyglucose (2-DG) uptake (a measure of glucose transport activity) by various types of skeletal muscle fibers both in vivo and in vitro (isolated muscle, cultured myoblasts) was diminished in diabetic GK rats. This diabetes-related suppression of skeletal muscle glucose utilization was associated with a decrease in insulin's ability to promote the phosphorylation of tyrosine residues of insulin receptor substrate-1 (IRS-1). Similarly, the translocation of GLUT-4 from intracellular compartment to plasma membrane in response to insulin was also reduced in these animals. Oxidative stress-based markers (e.g. urinary isoprostane, carbonyl-bound proteins) were elevated as a function of diabetes. Nullification of the heightened state of oxidative stress in the GK rats with alpha-lipoic acid resulted in a partial amelioration of the diabetes-related impairment of the in vivo and in vitro insulin actions. Collectively, the above data suggest that 1) insulin resistance in GK rats occurs at the hepatic and skeletal muscle levels, 2) muscle cell glucose transport exhibited a blunted response to insulin and it is associated with a major defect in key molecules of both GLUT-4 trafficking and insulin signaling pathways, 3) skeletal muscle insulin resistance in GK rats appears to be of genetic origin and not merely related to a paracrine or autocrine effect, since this phenomenon is also observed in cultured myoblasts over several passages and finally heightened state of oxidative stress may mediate the development of insulin resistance during diabetes.     

Discovery and preclinical development of AR453588 as an anti-diabetic glucokinase activator

Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high levels of glucose. Flux through GK is also responsible for reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can activate GK could provide a therapeutic benefit. Herein we report the further structure activity studies of a novel series of glucokinase activators (GKA). These studies led to the identification of pyridine 72 as a potent GKA that lowered post-prandial glucose in normal C57BL/6J mice, and after 14d dosing in ob/ob mice.     

Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.     

Long-term treatment with the Na+-glucose cotransporter inhibitor T-1095 causes sustained improvement in hyperglycemia and prevents diabetic neuropathy in Goto-Kakizaki Rats

We examined the effects of T-1095, an orally active inhibitor of Na(+)-glucose cotransporter (SGLT), on the development and severity of diabetes in Goto-Kakizaki (GK) rat, a spontaneous, non-obese model of type 2 diabetes. T-1095 was administered as dietary admixture (0.1% w/w) beginning at 7 weeks of age for 32 weeks. Untreated male GK rats were hyperglycemic compared with Wistar rats. Throughout the study, T-1095 treatment significantly decreased both blood glucose and hemoglobin A(1C) levels in the GK rats. The concomitant increase of urinary glucose excretion indicated that the hypoglycemic action of T-1095 is derived from the enhancement of urinary glucose disposal. Although food intake was not changed in the T-1095-treated rats, the body weight gain was retarded. T-1095 treatment partially ameliorated oral glucose tolerance but not the impaired glucose-induced insulin secretion. Homeostasis model assessment (HOMA) indicated the existence of insulin resistance in GK rats and a significant restoration by T-1095-treatment. There was a reduction of the thermal response in tail-flick testing following long-term hyperglycemia (diabetic neuropathy). Treatment of T-1095 significantly prevented the development of diabetic neuropathy in male GK rats. Sustained improvement of hyperglycemia and prevention of diabetic neuropathy by the T-1095-treatment provide further support the use of SGLT inhibitors for the treatment of diabetes.     

Effects of hexapeptide, a compound analogous to insulin B chain fragment B-21-26(DP-432) on the glucose uptake into the perfused hind limb of rats

DP-432 is a synthetic new peptide analogous to insulin B-chain fragment B21-26. It has been reported that this hexapeptide shows insulin potentiating action besides insulin-like activities in adipose tissue and diaphragm of rats. The perfusion of the hind limb of rats was performed according to the procedure of Ruderman with some modifications. The medium was Krebs-Ringer bicarbonate buffer containing 0.5% bovine albumin and 8.3 mM glucose. The erythrocytes were omitted from the medium. In this experimental procedure it was shown that the glucose uptake into the hind limb of rats increased by infusion of DP-432 (100 microgram/ml). The amount of glucose uptake into the hind limb responded to insulin doses (50, 100, 1000 muU/ml). DP-432 had insulin-like activity of 100 muU/ml. However, DP-432 itself did not have any potentiation of insulin activity on the muscle.     

The triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic-acid methyl ester has potent anti-diabetic effects in diet-induced diabetic mice and Lepr(db/db) mice

The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic-acid (CDDO) and its methyl ester (CDDO-Me) are undergoing clinical trials in cancer and leukemia therapy. Here we report that CDDO-Me ameliorates diabetes in high fat diet-fed type 2 diabetic mice and in Lepr(db/db) mice. CDDO-Me reduces proinflammatory cytokine expression in these animals. Oral CDDO-Me administration reduces total body fat, plasma triglyceride, and free fatty acid levels. It also improves glucose tolerance and insulin tolerance tests. Its potent glucose-lowering activity results from enhanced insulin action. Hyperinsulinemic-euglycemic clamp reveals an increased glucose infusion rate required to maintain euglycemia and showed a significant increase in muscle-specific insulin-stimulated glucose uptake (71% soleus, 58% gastrocnemius) and peripheral glucose clearance as documented by a 48% increase in glucose disposal rate. CDDO-Me activates AMP-activated protein kinase (AMPK) and via LKB1 activation in muscle and liver in vivo. Treatment of isolated hepatocytes with CDDO-Me directly stimulates AMPK activity and LKB1 phosphorylation and decreases acetyl-coA carboxylase activity; it also down-regulates lipogenic gene expression, suppresses gluconeogenesis, and increases glucose uptake. Inhibition of AMPK phosphorylation using compound C and lentiviral-mediated knockdown of AMPK completely blocks the CDDO-Me-induced effect on hepatocytes as well as C(2)C(12) cells. We conclude that the triterpenoid CDDO-Me has potent anti-diabetic action in diabetic mouse models that is mediated at least in part through AMPK activation. The in vivo anti-diabetogenic effects occur at a dose substantially lower than that used for anti-leukemia therapy. We suggest that CDDO-Me holds promise as a potential anti-diabetic agent.     

Modulation of glucokinase by glucose, small-molecule activator and glucokinase regulatory protein: steady-state kinetic and cell-based analysis

GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact. In the presence of a liver-specific GKA (compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by compound A. We show that GKRP (GK regulatory protein) is a slow tight-binding inhibitor of GK. Analysis of progress curves indicate that this inhibition is time dependent, with apparent initial and final Ki values being 113 and 12.8 nM respectively. When GK is pre-incubated with glucose and compound A, the inhibition observed by GKRP is time dependent, but independent of GKRP concentration, reflecting the GKA-controlled transition between closed and open GK conformations. These data are supported by cell-based imaging data from primary rat hepatocytes. This work characterizes the modulation of GK by a novel GKA that may enable the design of new and improved GKAs.     

Glucokinase as an emerging anti-diabetes target and recent progress in the development of its agonists

Type 2 diabetes mellitus is a metabolic disorder with complicated pathogenesis, and mono-target therapy often fails to effectively manage the levels of blood glucose. In recent years, the anti-diabetes target glucokinase (GK) has attracted the attention of researchers. It acts as a glucose sensor, triggering counter regulatory responses following a change in glucose levels to aid restoration of normoglycemia. Activation of GK induces glucose metabolism and reduces glucose levels for the treatment of type 2 diabetes. GK agonists (GKA) are a new class of antidiabetic drugs. Among these agents, dorzagliatin is currently being investigated in phase III clinical trials, while PB-201 and AZD-1656 have reached phase II clinical trials. This article describes the mechanism of action of GK in diabetes and of action of GKA at the protein level, and provides a review of the research, trends, and prospects regarding the use of GKA in this setting.