Masahiro Mizota

Mechanism of the lipid-lowering effect of ethyl all-cis-5,8,11,14,17-icosapentaenoate

The effect of highly purified ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on cholesterol metabolism in rats was examined to clarify the mechanism of its hypolipidemic action. Pretreatment with EPA-E reduced the increase in plasma radioactivity after oral administration of [14C]cholesterol. The conversion of [14C]3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) to [14C]mevalonic acid was significantly inhibited in liver microsomes obtained from rats treated with EPA-E. There was an increase in free cholesterol and a marked rise in the eicosapentaenoic acid (EPA) content of phospholipids in these microsomes. EPA-E restored the suppression of biliary secretion induced by feeding a casein-rich diet to bile duct-cannulated rats. Furthermore, when serum lipoprotein (d < 1.210) from rats given EPA-E was i.v. injected into normal rats, a more rapid elimination of cholesterol was observed as compared to that in rats injected with lipoprotein from EPA-E-untreated rats. This rapid clearance was found in the lipoprotein fractions of d < 1.006 and 1.006 < d < 1.063. These findings suggest that EPA-E has an inhibitory effect on intestinal cholesterol absorption and hepatic cholesterol biosynthesis, and an enhancing effect on hepatic biliary secretion. EPA-E would also seem to cause modification of serum lipoproteins, whereby their clearance from the serum is increased.

Ethyl all-cis-5,8,11,14,17-icosapentaenoate modifies the biochemical properties of rat very low-density lipoprotein

The biochemical properties of serum very low-density lipoprotein (VLDL) were investigated in rats given highly purified all-cis-5,8,11,14,17-icosapentaenoate (EPA), an ethyl-ester derivative (EPA-E). The elution time (gel filtration) of VLDL from EPA-E-treated serum was increased significantly compared with that of the control. EPA-E-treated VLDL isolated by ultracentrifugation exhibited a marked decrease in triglyceride content with a relative increase in cholesterol. In treated VLDL, a significant increase in the ratio of apo E/apo C was observed. There was a remarkable increase in the content of EPA in all the fractions of phospholipids, cholesteryl esters and triglycerides after EPA-E treatment, resulting in n-3 polyunsaturated fatty acid-rich VLDL. EPA-E also reduced the incorporation of [14C]oleate into triglycerides in hepatic microsomes and the rate of hepatic triglyceride secretion. Moreover, lipoprotein lipase activity in heparin-injected plasma was increased in rats given EPA-E without there being an effect on hepatic triglyceride lipase activity. These findings indicate that EPA-E exerts an inhibitory effect on hepatic triglyceride synthesis/secretion and a stimulatory effect on triglyceride degradation, resulting in a reduction in particle size and an increase in the ratio of apo E/apo C. Triglyceride-poor and EPA-rich VLDL may rapidly be converted into the density of intermediate low-density lipoprotein and low-density lipoprotein and/or may be absorbed into the liver rapidly.

Effects of ethyl all-cis-5,8,11,14,17-icosapentaenoate on the physical properties of arterial walls in high cholesterol diet-fed rabbits

The effects of ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on in vivo physical properties of arteriosclerotic aorta and femoral artery in high cholesterol diet (HCD)-fed rabbits were studied. The aortic pulse wave velocity (PWV) of rabbits fed HCD for 12 weeks (control group) tended to be higher than that of rabbits fed a normal diet (normal group). Because the PWVs in HCD-fed rabbits administered orally with 30 and 300 mg/kg/day EPA-E were significantly lower than the PWV of the control group, the distensibility of arteriosclerotic aorta was improved with administration of EPA-E. The stiffness parameter (β) value as an in vivo indicator of arteriosclerosis was significantly higher in the control group than in the normal group and improved with administration of EPA-E to almost the same level as that of the normal group. The β-values were in significant negative correlation with medial elastin content and medial smooth muscle cell (SMC) density in thoracic aorta and in positive correlation with the free cholesterol content in abdominal aortic SMC. On the other hand, they were not correlated with either the cross-sectional area of intimal thickening lesions or the plasma lipid levels measured simultaneously. The femoral PWVs were, like those in the aorta, higher in the control group as compared with the normal group, and the changes were improved with administration of EPA-E. These results show that EPA-E improved the in vivo distensibility of arteriosclerotic arteries in HCD-fed rabbits. The mechanism of this improvement with EPA-E appeared to be related to histologic amelioration in the aortic medial elastic fibers and SMC and to protection from free cholesterol accumulation in SMC as one of the probable causes.

5-hydroxydecanoate inhibits ATP-sensitive K+ channel currents in guinea-pig single ventricular myocytes

We investigated the effect of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the electrical activity of guinea-pig ventricular myocytes. The outward K+ current increased by lowering the intracellular ATP concentration (0.5 mM) was efficiently blocked by 5-hydroxydecanoate when recording in the whole cell configuration with the application of voltage ramps. The increase in the time-independent outward K+ current induced by reducing intracellular ATP to 0 mM was also blocked by 5-hydroxydecanoate (10 or 100 microM) and by tolbutamide (1 mM). Using the single channel recording technique, we found that 5-hydroxydecanoate blocked ATP-sensitive K+ channels when its channel open probability was increased by 1 mM ATP together with 1 mM ADP or by an intracellular pH of 6.6. These conditions are well documented to reflect metabolic changes in the early stages of myocardial ischemic attack. These results suggest that 5-hydroxydecanoate could inhibit ATP-sensitive K+ channels, resulting in an antiarrhythmic effect specifically on ischemic hearts.

Protective action of ulinastatin against cisplatin nephrotoxicity in mice and its effect on the lysosomal fragility.

The development of azotemia after cisplatin injection in mice was inhibited by ulinastatin treatment in a dose-dependent manner. Reduction in creatinine clearance and elevation in fractional excretion of sodium in mice receiving cisplatin was ameliorated by ulinastatin administration. Epithelial necrosis and hyaline cast formation in the proximal tubule were also suppressed. Ulinastatin showed no influence on the kidney platinum level after cisplatin injection. In LLC-PK1 cells, addition of ulinastatin to the incubation medium markedly reduced the release of N-acetyl-beta-D-glucosaminidase, on of the lysosomal enzymes, during hypotonic treatment only when cells were damaged with cisplatin. On the other hand, ulinastatin showed no effect on the elevation of malondialdehyde concentration in the murine kidney cortical slices after the treatment with cisplatin. These results indicate that ulinastatin has a protective effect against cisplatin nephrotoxicity, and its prevention of the increase in lysosomal fragility is a probable mechanism involved in the renal protection.

Amelioration of insulin resistance in genetically obese rodents by M16209, a new antidiabetic agent

Improvement of metabolic disorders by M16209 (1-(3-bromobenzofuran-2-ylsulfonyl)hydantoin), an antidiabetic agent, was studied in genetically obese Zucker fa/fa rats and C57BL/6J ob/ob mice. In fa/fa rats oral administration of M16209 (30 and 100 mg/kg/day) for 7 days dose dependently improved hyperinsulinemia without affecting body weight. Oral glucose loading (2 g glucose/kg body weight) after 10 days of administration to fa/fa rats revealed that M16209 significantly improved glucose tolerance both 30 and 60 min after glucose loading, but did not affect preload serum glucose levels. At one day after 13 days of administration of M16209, the serum levels of triglyceride, total cholesterol and free fatty acid were clearly lower in treated fa/fa rats than those in untreated rats. In C57BL/6J ob/ob mice, M16209 given for 28 days at doses of 30 and 100 mg/kg/day improved hyperinsulinemia, hyperglycemia and hypercholesterolemia without affecting body weight. In a hyperinsulinemic euglycemic clamp study in fa/fa rats, administration of M16209 for 7 days at a dose of 100 mg/kg/day significantly normalized the decreased metabolic clearance rate but did not show any effect on the augmented hepatic glucose output. These findings demonstrate that improvement of metabolic disorders in genetically obese rodents by M16209 is due to amelioration of insulin resistance in peripheral tissues.

Improvement of metabolic disorders and visceral fat obesity by the β3-adrenoceptor agonist (R*,R*)-(±)-methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl]-phenoxyacetate hydrobromide (BRL35135A) in genetically obese rodents

The effects of BRL35135A ((R*,R*)-(+/-)-methyl-4-[2-[2-hydroxy-2 -(3-chlorophenyl)ethylamino]propyl]-phenoxyacetate hydrobromide), a beta 3-adrenoceptor agonist, on visceral and subcutaneous fat weight and metabolic disorders were studied in genetically obese C57BL/KsJ db/db mice and Zucker fa/fa rats. In db/db mice, four weeks of oral administration of BRL35135A (0.5 and 5 mg/kg/day) decreased body weight gain and reduced white fat weight. The rates of reduction of white fat weight were in the order mesenteric fat > retroperitoneal fat > subcutaneous fat. In fa/fa rats, daily administration of BRL35135A (0.05 mg/kg/day)) for 6 weeks reduced the visceral white fat weight/total energy intake ratio, particularly for mesenteric fat, without any clear effect on body weight gain. This tendency of the compound to exert effects on visceral fat was consistent with the findings that the effect of BRL37344 ((R*,R*)-(+/-) -methyl-4-[2-[2-hydroxy-2-(3-chlorophenyl)ethylamino]propyl]-phenoxyacet ic acid), an active metabolite of BRL35135A, on the lipolytic activity of isolated adipocytes and the tissue concentration of [14C]BRL37344 in male Wistar rats were each greater in visceral fat than in subcutaneous fat. Moreover, BRL35135A at 0.05 mg/kg/day elevated serum insulin levels and improved hyperglycemia in db/db mice without reducing body weight gain, whereas at doses of 0.5 and 5 mg/kg/day it ameliorated hyperglycemia and hyperlipidemia, and tended to decrease serum insulin levels. In fa/fa rats, BRL35135A (0.005 mg/kg/day) was also effective in improving hyperinsulinemia, glucose intolerance, and hypertriglyceridemia without any effect on body weight gain or fat distribution. These findings suggest that the improvement of metabolic disorders by BRL35135A may be due to improvement in insulin resistance as well as reduction of visceral fat weight.

A cAMP-dependent protein kinase inhibitor modulates the blocking action of ATP and 5-hydroxydecanoate on the ATP-sensitive K+ channel.

We studied the blocking mechanism of 5-hydroxydecanoate, a novel antiarrhythmic agent, on the ATP-sensitive K+ channel in the single ventricular myocytes using the inside-out patch clamp technique. The channel activity in response to 5-hydroxydecanoate varied with each membrane patch corresponding to the sensitivity to ATP. In this condition the exogenous application of cAMP or cAMP-dependent protein kinase (PKA) obviously recovered the ATP-sensitive K+ channel activity after channel deactivation. By contrast, in membrane patches exhibited low sensitivity to ATP, endogenous cAMP-dependent protein kinase inhibitor (PKI) depressed the channel activity and restored the inhibitory action of 5-hydroxydecanoate and ATP on the channel. These results suggest that PKA-PKI system is involved in the regulatory mechanism of gating activity of the ATP-sensitive K+ channel and the blocking action of 5-hydroxydecanoate and ATP appears to be exerted by potentiating the inhibitory action of PKI on the channel.

Effects of novel aldose reductase inhibitors, M16209 and M16287, on streptozotocin-induced diabetic neuropathy in rats

We investigated the effects of novel aldose reductase inhibitors, M16209 (1-(3-bromobenzo[b]furan-2-ylsulfonyl)hydantoin) and M16287 (1-(3-chlorobenzo[b]furan-2-ylsulfonyl)hydantoin), on neuropathy in streptozotocin-induced (STZ) diabetic rats. Both compounds (3-100 mg/kg per day, p.o.) dose dependently improved the decreased motor nerve conduction velocity in the sciatic nerve during a 14-day treatment period. These compounds also partially ameliorated the diabetes-induced histological changes in the sciatic nerve. A distinct increase in sorbitol content and a slight decrease in myo-inositol content was observed in the sciatic nerve of STZ diabetic rats, and the sorbitol accumulation was dose dependently suppressed by treatment with M16209 and M16287. Treatment started at an earlier period was more effective in the suppression of sorbitol accumulation. There was a significant correlation between motor nerve conduction velocity and nerve sorbitol content, whereas there was none between motor nerve conduction velocity and myo-inositol content. The present study indicates that M16209 and M16287 are potent aldose reductase inhibitors expected to be useful for the treatment of diabetic complications.

A novel quinolinone diuretic, M12285, and its activation mechanism through sulfate conjugation.

The diuretic activity of a quinolinone oxime diuretic, M12285, was examined after renal arterial, i.v. and portal injection in rats. M12285 injected into the renal artery at a dose of 1 mg/kg caused no diuretic effect, whereas i.v. and portal injections induced marked diuresis dose dependently. The minimum effective dose with portal injection was lower (1 mg/kg) than that with i.v. injection (3 mg/kg) and the start of the effect was faster with portal injection. These results indicated that some metabolic modification in the liver is necessary for the diuretic activity to appear. Accordingly, we performed in situ rat liver perfusion with M12285 and obtained several metabolites. Renal arterial injection of each fractionated metabolite of M12285 revealed that all the diuretic activity derived from one of these metabolites. From IR and 1H-nuclear magnetic resonance (1HNMR) measurements, the chemical structure of this active metabolite was assumed to be a sulfate-conjugated form of M12285 at the oxime moiety. Based on this tentative chemical structure, we synthesized the oxime sulfate of M12285 (potassium salt, M17000) and confirmed the identity of IR and 1HNMR spectra. Administration of M17000 into the renal artery induced apparent diuresis in a dose-dependent manner in both rats and dogs. These results indicate that the oxime sulfate of M12285 is responsible for the diuretic activity of M12285. Therefore, we synthesized several derivatives of M17000 and confirmed their possible therapeutic value as a novel family of diuretics, namely quinolinone oxime sulfonic acids.