Lipogenic action of the novel oral antidiabetic agent HQL-975 in genetically obese diabetic KK-Ay mice.

HQL-975 (3-{4-12-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxyl-phenyl}-2S- propylamino-propionic acid) is a new oral antidiabetic agent which has been shown to be effective in insulin-resistant diabetic animals. In the present study, we examined the effects of HQL-975 on glucose utilization and insulin action in KK-Ay mice with genetically obese non-insulin diabetes. (1) Dietary administration of HQL-975 (19 mg/kg/d for 7 d) improved hyperglycemia, hyperlipidemia and hyperinsulinemia in the mice. (2) The HQL-975-treated mice showed enhanced net glucose utilization, that is, glucose was significantly incorporated into total lipids in the white adipose tissue (WAT) and liver, and into glycogen in the diaphragm for the last 24 h of the drug administration period. (3) Treatment improved the decreased stimulative action of insulin in the epididymal WAT and the agent increased insulin-stimulated lipogenesis from both glucose and acetate. (4) Treatment also increased the activity of lipogenic enzymes such as glycerol-3-phosphate dehydrogenase and fatty acid synthetase. (5) In vitro exposure of WAT to HQL-975 enhanced lipogenesis in the presence of insulin. From these findings, we conclude that HQL-975 improves glucose utilization of KK-Ay mice through the enhancement of insulin action, which is associated with its lipogenic effects.

Selection of an RNA molecule that specifically inhibits the protease activity of subtilisin.

RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA by SELEX (systematic evolution of ligands by exponential enrichment) and by use of a subtilisin-immobilized Sepharose column. After eight rounds of selection, RNA aptamers were isolated by cloning to a plasmid vector. We characterized one of the selected RNA molecules. This RNA aptamer displayed specific inhibition toward the subtilisin activity, even when the assay for subtilisin was performed using the chromogenic small peptide as substrate, and almost no inhibitory activity toward trypsin and chymotrypsin, although these enzymes are serine proteases similar to subtilisin. These findings indicate that this RNA can differentially recognize the surfaces of similar proteases. Kinetic analysis of the RNA aptamer revealed that the inhibition constant (Ki) toward subtilisin was 2.5 microM.

Actions of the novel oral antidiabetic agent HQL-975 in genetically obese diabetic db/db mice.

The hypoglycemic effect of the novel oral agent 3-[4-12-(5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]-phenyl]-2S-propyla mino-propionic acid (HQL-975) was examined in db/db mice with genetically obese non-insulin dependent diabetes mellitus (NIDDM). The oral administration of HQL-975 at 3.5 and 35.3 mg/kg/d for 7 d decreased the plasma glucose level of these mice in a dose-dependent manner. HQL-975 also significantly decreased the plasma triglyceride, total cholesterol, non-esterified fatty acid and insulin levels. In the oral glucose tolerance test, HQL-975-treated mice showed improved glucose tolerance and decreased endogenous insulin secretion. HQL-975 increased glycemic response to exogenous insulin in the mice. In the HQL-975-treated db/db mice adipocytes, the glucose uptake, insulin binding, and GLUT4 expression were increased compared with those in untreated db/db mice adipocytes. These results indicate that HQL-975 improved insulin action in db/db mice through receptor and post-receptor effects. In conclusion, HQL-975 is a new oral antidiabetic agent with a hypoglycemic effect which is associated with an insulin-sensitizing effect. This agent may therefore be effective for the treatment of NIDDM.

Actions of the novel oral antidiabetic agent HQL-975 in insulin-resistant non-insulin-dependent diabetes mellitus model animals.

The hypoglycemic effects of a novel oral antidiabetic agent HQL-975, were studied in normal rats, streptozotocin-induced diabetic (STZD) rats and genetically insulin-resistant non-insulin-dependent diabetes mellitus (NIDDM) model animals, KK-Ay mice and Zucker diabetic fatty (ZDF) rats. After the dietary administration of HQL-975 to KK-Ay mice, significant decreases in plasma glucose, insulin, triglyceride and non-esterified fatty acid levels were observed. The effective dosage of HQL-975 to decrease the plasma glucose level by 30% was 3.1 mg/kg per day. However, the plasma glucose level was not altered after the administration of HQL-975 in normal and STZD rats. The results suggest that HQL-975 is more effective against the abnormalities of glucose and lipid metabolism of insulin-resistant model animals than in that of normal and insulin-deficient diabetic animals. It is reported that ZDF rats indicate a severely diabetic state as a result of insulin resistance and further the presence of beta-cell insulin secretory defects. Here, HQL-975 (1-30 mg/kg per day for 7 days) was administered to ZDF rats; slight decreases in the plasma glucose (18%) and lipids (41%) levels were observed in the rats given 30 mg/kg. To clarify the action mechanism of HQL-975, we studied the effects of HQL-975 administration on the insulin action of target tissues in KK-Ay mice. After the dietary administration of HQL-975 (0.001, 0.003, 0.010% for 7 days) to KK-Ay mice, hepatic glycolytic and gluconeogenic key enzyme activities were measured. The glucose 6-phosphatase activity was decreased (20-40%) as compared with control. The results suggest that HQL-975 enhances the insulin action in hepatic enzyme regulation. To investigate the actions of HQL-975 in peripheral tissues such as muscle and adipose, an in vivo glucose uptake study using 3H-2-deoxyglucose was performed in KK-Ay mice treated with HQL-975 (0.010% for 7 days). The 2-deoxyglucose uptake of the basal state was not altered, but the insulin-stimulated 2-deoxyglucose uptake in muscle (41-191%) and adipose (46-88%) tissues was increased by the HQL-975 treatment as compared with control. These results suggest that HQL-975 also enhances the insulin action of peripheral tissues. Based on these findings, HQL-975 is expected to be useful for treatment of insulin-resistant patients with NIDDM.

Effects of the novel oral antidiabetic agent HQL-975 on glucose and lipid metabolism in diabetic db/db mice.

The antidiabetic effects of 3-¿4-[2-(5-methyl-2-phenyl-oxazol-4- yl)ethoxy]phenyl¿-2S-propylamino-propionic acid (CAS 185679-16-7, HQL-975), a novel oral agent, on a genetically obese non-insulin-dependent diabetes mellitus (NIDDM) model (db/db mice) were examined. HQL-975 administration (3.7-34.1 mg/kg/d for 7 days) decreased the levels of plasma glucose, triglyceride, total cholesterol, non-esterified fatty acid and insulin in the mice. In an intraperitoneal glucose tolerance test (IPGTT), HQL-975 administration decreased the fasting plasma glucose level and improved the glucose tolerance in the mice. The HQL-975 administration also significantly increased the glycogenesis and lipogenesis from 14C-glucose in liver, but did not alter the glycogenesis in the diaphragm or the lipogenesis in adipose tissues at 2 h after the glucose loading. In the HQL-975-treated db/db mice, the radioactivity of 14C-glucose incorporated into hepatic glycogen was higher than that incorporated into hepatic total lipids. After the administration of HQL-975 (34.1 mg/kg/d for 7 days) to db/db mice, the hepatic hexokinase and fatty acid synthetase activities were significantly increased, the glycogen synthase I activity was increased but not significantly, and the glucose-6-phosphatase and the phosphoenolpyruvate carboxykinase activities were decreased. These results suggest that HQL-975 increases the hepatic glucose utilization and decreases the hepatic glucose production. Since hepatic glycogenesis is regulated by glucose itself but not by insulin in normoglycemic ICR mice, HQL-975 is thought to enhance the effect of glucose on the stimulation of hepatic glycogenesis. It is concluded that the enhancement of the hepatic glucose utilization played an important role in the hypoglycemic action of HQL-975.

RNA aptamers to a protease, subtilisin.

RNA ligands (RNA aptamers) to a protease subtilisin were selected from pools of random RNA using SELEX (Systematic evolution of ligands by exponential enrichment). We selected RNA aptamers using a subtilisin-immobilized Sepharose column. After several rounds of selection, we obtained RNA aptamers that specifically inhibit the subtilisin activity. Characterization of the RNA aptamers will be reported.

Effect of neonatal thymectomy on experimental autoimmune hepatitis in mice.

Using an autoimmune hepatitis model of A/J mice which was prepared with immunization by syngeneic crude liver proteins, various influences of neonatal thymectomy were studied by observations of histological liver changes, autoantibody to liver-specific membrane lipoprotein (LSP), delayed-type hypersensitivity (DTH) to LSP, and purified protein derivative (PPD), and suppressor activity to LSP. The liver changes in the thymectomized mice were more intense than those in the non-thymectomized controls. Production of the anti-LSP autoantibodies and positive DTH to syngeneic LSP could be recognized in both groups of the thymectomized mice and the non-thymectomized controls, but the levels of those were higher in the former. In the level of DTH to PPD the thymectomized mice were lower than the non-thymectomized controls. Adoptive transfer experiments showed that suppressor activity to LSP was reduced in the spleen cells of neonatally thymectomized mice. This experiment suggests that neonatal thymectomy is apt to abolish tolerance to LSP on account of depressed suppressor activity to autoantigen, and accordingly liver damage is increased.

Studies on a new immunoactive peptide, FK-156. IV. Synthesis of FK-156 and its geometric isomer.

For the structural confirmation of FK-156, two possible structures, 1 and its geometric isomer 2, were synthesized. Di-Z-meso-diaminopimelic acid (4) was converted into 14 via a sequence of reactions involving, as key steps, an enzyme-mediated asymmetric hydrolysis (6 leads to 7), followed by carbobenzyloxylation using a copper chelate procedure (7 leads to 8). Condensation of 14 and the appropriately protected lactoyl dipeptide 17 and removal of the protecting groups of the resulting 18 afforded 1. Protection of 7 to 22, followed by coupling to glycine via an azide method, gave 25. Derivatization of 25 to 29 and condensation with 17 gave 30, which was deprotected to yield 2. Compound 1 proved to be identical in all respects with the natural product.