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...

We addressed the question of whether both mitochondrial and cytoplasmic translation activities decreased simultaneously in human skin fibroblasts with the age of the donors and found that the age-related reduction was limited to mitochondrial translation. Then, to determine which genome, mitochondrial or nuclear, was responsible for this age-related, mitochondria-specific reduction, pure nuclear transfer was carried out from mitochondrial DNA (mtDNA)-less HeLa cells to four fibroblast lines, two from aged subjects, one from a fetus, and one from a patient with cardiomyopathy, and their nuclear hybrid clones were isolated. A normal fibroblast line from the fetus and a respiration-deficient fibroblast line from the patient were used as a positive and a negative control, respectively. Subsequently, the mitochondrial translation and respiration properties of the nuclear hybrid clones were compared. A negative control experiment showed that this procedure could be used to isolate even nuclear hybrids expressing overall mitochondrial respiration deficiency, whereas no respiration deficiencies were observed in any nuclear hybrids irrespective of whether their mtDNAs were exclusively derived from aged or fetal donors. These observations suggest that nuclear-recessive mutations of factors involved in mitochondrial translation but not mtDNA mutations are responsible for age-related respiration deficiency of human fibroblasts.It has been presumed that somatic mutations accumulate in mitochondrial DNA (mtDNA) much faster than in nuclear DNA because mitochondria are highly oxygenic organelles due to their function in producing energy, mtDNA lacks histones protecting it from mutagenic damage, and its repair systems are limited (1). Therefore, it has been proposed that the accumulation of various somatic mutations in mtDNA and the resultant decrease in mitochondrial respiratory function could be involved in aging processes in mammals (2-4). There have been many reports that the respiration capacity of mitochondria in highly oxidative tissues decreases during aging (4). Moreover, the accumulation of somatic and pathogenic mtDNA mutations, which have been shown to cause various kinds of mitochondrial encephalomyopathies (5-8), was also shown to increase with age in normal subjects (9, 10). However, as the nuclear genome encodes most mitochondrial proteins including factors necessary for replication and expression of the mitochondrial genome, it is possible that only mutations in the nuclear genome contribute to the age-related decline of mitochondrial respiratory function. In fact, there is no convincing evidence that mtDNA somatic mutations are responsible for this age-related phenotype.Previously, we observed age-related reduction of cytochrome c oxidase (COX) 1 activity and mitochondrial translation in cultured human skin fibroblasts isolated from donors of various ages (0 -97 years), and in studies on their mtDNA transfer to mtDNA-less ( 0 ) HeLa cells, we showed that mtDNA mutations were not responsible for the observed age...

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Glucokinase activators (GKAs) are small-molecule agents that enhance glucose sensing by pancreatic ␤ cells and glucose metabolism by hepatocytes. There is strong interest in these agents as potential therapies for type 2 diabetes. Here, we report key pharmacokinetic and pharmacodynamic findings from preclinical studies of the GKA 3- [[6-(ethylsulfonyl. Incubated in vitro with recombinant human glucokinase, 1 M MK-0941 lowered the S 0.5 of this enzyme for glucose from 6.9 to 1.4 mM and increased the maximum velocity of glucose phosphorylation by 1.5-fold. In 2.5 and 10 mM glucose, the EC 50 values for activation of GK by MK-0941 were 0.240 and 0.065 M, respectively. Treatment of isolated rat islets of Langerhans and hepatocytes with 10 M MK-0941 increased insulin secretion by 17-fold and glucose uptake up to 18-fold, respectively. MK-0941 exhibited strong glucose-lowering activity in C57BL/6J mice maintained on a high-fat diet (HFD), db/db mice, HFD plus low-dose streptozotocin-treated mice, and nondiabetic dogs. In both mice and dogs, oral doses of MK-0941 were rapidly absorbed and rapidly cleared from the blood; plasma levels reached maximum within 1 h and fell thereafter with a half-life of ϳ2 h. During oral glucose tolerance testing in dogs, MK-0941 reduced total area-under-the-curve postchallenge (0 -2 h) plasma glucose levels by up to 48% compared with vehicletreated controls. When administered twice daily to mice for 16 days, and once daily to the dog for 4 days, MK-0941 remained efficacious on successive days. These findings support further investigation of MK-0941 as a potential therapeutic agent for treatment of type 2 diabetes.

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