Teruhiko Shimokawa

Discovery of Novel Forkhead Box O1 Inhibitors for Treating Type 2 Diabetes: Improvement of Fasting Glycemia in Diabetic db/db Mice

Excessive hepatic glucose production through the gluconeogenesis pathway is partially responsible for the elevated glucose levels observed in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box O1 (Foxo1) plays a crucial role in mediating the effect of insulin on hepatic gluconeogenesis. Here, using a db/db mouse model, we demonstrate the effectiveness of Foxo1 inhibitor, an orally active small-molecule compound, as a therapeutic drug for treating T2DM. Using mass spectrometric affinity screening, we discovered a series of compounds that bind to Foxo1, identifying among them the compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), which potently inhibits human Foxo1 transactivation and reduces glucose production through the inhibition of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase mRNA levels in a rat hepatic cell line. Oral administration of AS1842856 to diabetic db/db mice led to a drastic decrease in fasting plasma glucose level via the inhibition of hepatic gluconeogenic genes, whereas administration to normal mice had no effect on the fasting plasma glucose level. Treatment with AS1842856 also suppressed an increase in plasma glucose level caused by pyruvate injection in both normal and db/db mice. Taken together, these findings indicate that the Foxo1 inhibitor represents a new class of drugs for use in treating T2DM.

Expression of prostaglandin endoperoxide synthase-1 in a baculovirus system

A cDNA coding for ovine prostaglandin endoperoxide (PGH) synthase-1 was used to construct a recombinant baculovirus which was expressed in Spodoptera frugiperda (Sf9) insect cells. Two proteins reactive with anti-PGH synthase antibody were produced. A larger protein (Mr = 72,000) coelectrophoresed with native enzyme; a smaller, more abundant protein (Mr = 66,000) was unglycosylated enzyme. About 90% of both the immunoreactivity and the cyclooxygenase activity were present in a low speed (10(5) g x min) pellet; variable but low peroxidase activities were observed in this fraction. The specific cyclooxygenase activity of solubilized PGH synthase-1 from Sf9 cells was 56 units/mg versus 112 units/mg for the same cDNA expressed in cos-1 cells. The baculovirus-insect cell system is not ideal for generating large amounts of active PGH synthase-1 apparently because of inefficient N-glycosylation.

Effect of YM-126414 on glucose uptake and redistribution of glucose transporter isotype 4 in muscle cells

We discovered a novel compound, YM-126414 [1,3, 3-trimethyl-2-(2-phenylaminovinyl)-3H-indolium perchlorate], which stimulates glucose uptake in skeletal muscle cells in vitro. This compound increased the rate of consumption of glucose by C2C12 mouse myoblast cells in a dose-dependent manner (EC(50)=10 nM). To investigate the mechanism of this stimulation, we determined the redistribution of insulin-regulatable glucose transporter isotype 4 (Glut4). When fully differentiated C2C12 cells stably expressing myc-tagged Glut4 protein were treated with YM-126414, redistribution was dramatically increased in a dose-dependent manner (EC(50)=21 nM). These results indicate that YM-126414 is a novel glucose uptake stimulator for muscle cells by causing up-regulation of Glut4 redistribution in differentiated muscle cells. Our findings for the in vitro effects of YM-126414 suggest a direction for the development of new drugs for the treatment of type 2 diabetes.

Glucose uptake stimulator YM‐138552 activates gene expression and translocation of glucose transporter isotype 4

In this study, we examined the effect of YM‐138552 on the glucose uptake, gene expression, and transport activities of the insulin‐regulatable glucose transporter isotype 4 (Glut4) in skeletal muscle cells. YM‐138552 stimulated medium glucose consumption in a dose‐dependent manner (EC50 = 0.07 μM) in myoblast muscle C2C12 cells under differentiation conditions with 2% horse serum supplement. The stimulatory effect of glucose consumption was verified by radiolabeled 2‐DG uptake assay. The compound showed dose‐dependent stimulation of 2‐DG uptake in G8 myoblast muscle cells up to a 1 μM concentration (EC50 = 0.19 μM). To investigate the mechanism of glucose uptake stimulation by YM‐138552, the mRNA level of Glut4 was determined using real‐time quantitative RT‐PCR. The Glut4 mRNA level expressed in C2C12 cells treated with YM‐138552 increased up to at least 24 h (227% vs. control), after which it gradually decreased to the initial level at 36 h. In addition, we established that C2C12 cells stably expressed the myc‐tagged Glut4 protein (C2C12‐Glut4myc) and measured the Glut4 translocation activity. The Glut4 translocation activity was stimulated by treatment of YM‐138552 without insulin in a dose‐dependent manner (EC50 = 0.62 μM), and no insulin effect (100 nM) was observed. This suggests that YM‐138552 has an insulin‐like effect. These results suggest that the stimulation of glucose uptake by YM‐138552 in muscle cells was partly due to upregulation of the Glut4 gene expression and its translocation activation. Our findings on the in vitro effects of YM‐138552 glucose uptake stimulation through the Glut4 transporter may suggest a direction for the development of new drugs for the treatment of NIDDM. Drug Dev. Res. 51:43–48, 2000.

Amino Acids Essential to Catalysis by Prostaglandin Endoperoxide Synthase

The recent availability of cDNAs containing the entire coding region of prostaglandin endoperoxide (PGH) synthase (1–3) and the development of constructs which can be used for expressing the enzyme in vitro (4–6) permits the use of site-directed mutagenesis to characterize this enzyme (4–7). It is now possible to compare properties of mutagenized PGH synthases to the native enzyme in order to determine the effects of modifying any amino acid on the cyclooxygenase and/or hydroperoxidase activities, the size and spectral properties of the protein, and subtle changes in kinetic constants. Accordingly, we have applied mutagenesis procedures to identify amino acids which are important in cyclooxygenase and hydroperoxidase catalysis, heme binding, and the interaction of PGH synthase with nonsteroidal anti-inflammatory drugs. Here we present a summary of these studies in the context of developing a model for the cyclooxygenase and peroxidase active sites of the enzyme.