K. Shirai

Studies on cholesterol esterase in rat arterial wall

Cholesterol esterase activity was estimated in homogenates of rat arterial wall using radioactive cholesteryl oleate incorporated into phospholipid vesicles as a substrate. The labeled oleic acid was separated from the ester by addition of benzene-chloroform-methanol mixture. Under these conditions, two pH optima were found at about 4.5 and 7.5. Most of the activities at pH 4.5 and 7.5 were found in the lysosomal and microsomal fraction, respectively. No enzyme activity was detected when the substrate vesicles were prepared with phosphatidylethanolamine or sphingomyelin, but the activity was higher when the substrate vesicles were prepared with phosphatidylserine and highest when they were prepared with phosphatidylcholine. The relationship between enzyme regulation and lipid deposition in the arterial wall is discussed.

Lipoprotein lipase with a defect in lipid interface recognition in a case with type I hyperlipidaemia

Abstract. Defective lipoprotein lipase (LpL) was found in the postheparin plasma (PHP) of a patient with severe hypertriglyceridaemia. The patient was a 14‐year‐old girl with a maximum plasma triglyceride (TG) level of 3600 mg d‐1 who had been suffering from recurrent pancreatitis. The patients LpL purified from the PHP by heparin‐Sepharose and phenyl‐Sepharose chromatographies hydrolysed tributryrin, but not triolein emulsified with Triton X‐100 and phosphatidylcholine (PC), or in chylomicrons, whereas normal LpL hydrolysed these substrates. Moreover, unlike normal LpL, LpL from the patient did not associate with VLDL, as shown by Sepharose 4B column chromatography. The patients LpL hydrolysed triolein emulsified with lysophospholipid at a normal rate in the presence of apolipoprotein CII. These findings suggest that this patient has LpL with a normal catalytic site for tributyrin but with a defect in lipid interface recognition resulting in loss of ability to recognize VLDL or chylomicrons, but not of triolein emulsified with lysophospholipid.

Response of lipoprotein lipase to calorie intake in streptozotocin-induced diabetic rats

The mechanism regulating lipoprotein lipase (LPL) expression in adipose tissue was examined in rats in the conditions of different calorie intakes with and without streptozotocin-induced (STZ-) diabetes. The LPL activity released from adipose tissue was greater with the higher calorie intake (20 g of normal chow diet per day) than with the lower calorie intake (13 g of normal chow diet per day), and was greater in normal rats than in STZ-diabetic rats. The LPL activity was proportional to the serum insulin level in all conditions. Dot-blot analysis showed that the amount of LPL mRNA in adipose tissue was increased by the higher calorie diet and that the increase was less in the diabetic state. Expression of mRNA was also nearly parallel with the serum insulin level. LPL activity released from the heart was not affected by either the calorie intake or the diabetic state. These results suggest that the mechanisms of LPL expression in adipose tissue and the heart are different, and that LPL expression in adipose tissue was closely dependent on the insulin level.

Presence and Properties of Acyl Coenzyme A Synthetase for Medium-Chain Fatty Acids in Rat Intestinal Mucosa

A system was developed for assay of acyl coenzyme A (acyl CoA) activity on medium-chain fatty acids in rat intestinal mucosa. Using this system, we compared the characteristics of octanoyl (C8:0) CoA synthetase activity and palmitoyl (C16:0) CoA synthetase activity. Palmitoyl CoA synthetase activity as a function of palmitate concentration followed Michaelis-Menten kinetics, but octanoyl CoA synthetase activity as a function of octanoate concentration showed a biphasic reaction curve. The distributions of octanoyl CoA synthetase activity and palmitoyl CoA synthetase activity along the gastrointestinal tract were similar, both activities being present mainly in the middle portion of the small intestine. Incubation of octanoate with a homogenate of intestinal mucosa revealed that octnoate, like palmitate, is incorporated into phospholipids and triglycerides after its CoA activation. Oral administration of medium chain triglycerides induced a nearly 2-fold increase in octanoyl CoA synthetase activity in rat intestinal mucosa, whereas oral administration of long chain triglycerides did not affect the palmitoyl CoA synthetase activity. These results indicate that acyl CoA synthetase for medium-chain fatty acids in rat intestinal mucosa plays a key role in the utilization of medium-chain fatty acids for lipid synthesis, and that it is regulated in a different manner from acyl CoA synthetase for long-chain fatty acids.

Pseudohomozygous type II hyperlipoproteinemia

Nodular xanthomas on both elbows and a streak-like xanthoma on the intergluteal area developed in a 4-year-old girl with type IIa hyperlipoproteinemia. She had no disease associated with secondary hypercholesterolemia and no family history of hypercholesterolemia. Her xanthomas regressed under fat restriction diet and cholestyramine therapy. She was diagnosed as having pseudohomozygous type II hyperlipoproteinemia. The low-density lipoprotein (LDL) receptor activities of her cultured fibroblasts in terms of binding, internalization and degradation rate of LDL were normal. These results are consistent with a new syndrome of pseudohomozygous type II hyperlipoproteinemia and suggest that the mechanism of hypercholesterolemia, which induced xanthoma, differs from familial hypercholesterolemia.

Proliferation and triglyceride synthesizing activities of fibroblast-like cells derived from epididymal and subcutaneous adipose tissues of rats

Fibroblast-like cells from subcutaneous adipose tissue and epididymal adipose tissue were prepared from rats and their proliferation and [14C]deoxy-glucose uptake and triglyceride synthesis from [14C]palmitate were investigated. The proliferation of fibroblast-like cells from subcutaneous adipose tissue was greater than that from epididymal adipose tissue. Insulin enhanced the proliferation of fibroblast-like cells derived from subcutaneous adipose tissue, but not from epididymal adipose tissue. The uptake of [14C]deoxy-glucose by fibroblast-like cells from the two sources was similar. Triglyceride synthesis from [14C]palmitate by epididymal fibroblast-like cells was higher than that by subcutaneous fibroblast-like cells. The [14C]deoxy-glucose uptake and triglyceride synthesis from [14C]palmitate by fibroblast-like cells from the two sources were not enhanced by the addition of insulin. These findings suggest the existence of different types of adipocytes in subcutaneous and epididymal adipose tissues in terms of proliferation, and show that the proliferation of fibroblast-like cells from subcutaneous adipose tissue is regulated by insulin. The triglyceride synthesis by immature adipocytes from both adipose tissues was not affected by insulin.

Slowß-migrating lipoprotein: An atherogenic subclass of low density lipoproteins

Abstract Objective: To clarify the possibility that midband Lp in LDL fractions might act as an atherogenic lipoprotein in their interaction with macrophages. Design and Methods: Low density lipoproteins (LDL) isolated by zonal ultracentrifugation from midband lipoprotein-positive serum in type IIb hyperlipidemics were subjected to polyacrylamide gel disc electrophoresis. Results: A part of midband lipoprotein was observed between preβ- and β-band, in addition to the main β-band. We named this midband lipoprotein “slowβ-migrating Lp (slowβ-Lp).” The larger LDL subtraction from midband-lipoprotein positive serum on Sepharose 2B column chromatography contained much slowβ-Lp, named slowβ-Lp-rich LDL. The smaller LDL subfraction contained a little slowβ-Lp, named slowβ-Lp-poor LDL. Slowβ-Lp-rich LDL had similar composition to the control LDL except for apolipoprotein E. The uptake of [ 3 H]cholesteryl linoleate-labeled slowβ-Lp-rich LDL by J774 macrophages was higher than that of control LDL. The cholesterol ester content of J774 macrophages incubated with slowβ-Lp-rich LDL increased significantly compared with slowβ-Lp-poor LDL, β-VLDL, and control LDL. Conclusion: These results suggest that slowβ-Lp- in type IIb might generate foam cells from macrophages in atherosclerotic lesions.

Effect of 5-year administration of probucol on development of myocardial infarction in heterozygous familial hypercholesterolemia

Abstract This study was carried out to determine whether the decrease in plasma cholesterol produced by 5-year probucol administration would prevent development of myocardial infarction in patients with heterozygous familial hypercholesterolemia. Treated and untreated (control) groups included 39 and 91 patients, respectively. There were no differences in initial levels of total cholesterol (TC), low-density-lipoprotein (LDL)-cholesterol or high-density-lipoprotein (HDL)-cholesterol between groups. There were also no differences in age or incidence of risk factors at the beginning of the study. Probucol decreased levels of TC, LDL-cholesterol, and HDL-cholesterol by 23%, 24%, and 21%, respectively. Mean Achilles tendon thickening decreased from a median of 10.0 mm (range, 5.0 to 25.0 mm) to a median of 8.0 mm (range, 4.0 to 15.0 mm) over 5 years. Master double tolerance tests changed from positive to negative in five of 39 patients, and no patient experienced deterioration. There was no new episode of myocardial infarction in the treated group. Three cases of myocardial infarction were observed in the control group during the study. Results suggest that probucol administration might prevent development of myocardial infarction in patients with familial hypercholesterolemia.

Positive correlation between probucol in low density lipoprotein and LDL-lowering

SummaryThe relationship between the content of probucol in plasma and lipid lowering was studied after administration of probucol. The study group consisted of 44 patients with Type II hyperlipidaemia (mean age 56 y).Total cholesterol level was decreased from 293 to 232 mg · dl−1 by the administration of probucol. The plasma probucol concentration was 2.03 mg · dl−1 after 4 weeks of administration of 500 mg b. d. Although there was no relationship between the change in total cholesterol and the plasma probucol, a linerar relation was observed between the decrease in LDL-cholesterol concentration and the probucol concentration in LDL. Neither the VLDL nor the HDL-probucol content were related to the magnitude of the decrease in lipoprotein cholesterol.The present study has demonstrated that an increase in the probucol content in LDL was associated with a decrease in plasma LDL-cholesterol.

Effect of conditioning of β-migrating very low-density lipoprotein with macrophages on the accumulation of cholesteryl esters in smooth muscle cells

The mechanism of cholesteryl ester accumulation in smooth muscle cells was investigated. Incubation of smooth muscle cells with β-migrating very low-density lipoprotein (β-VLDL, d < 1.006) for 24 h did not result in accumulation of oil red O-stained particles in the cells. However, incubation of smooth muscle cells with β-VLDL in the presence of rat peritoneal macrophages induced accumulation of oil red O-stained granules in smooth muscle cells. Medium containing [3H]-cholesteryl linoleate-labelled β-VLDL ([3H]β-VLDL) that was conditioned with rat peritoneal macrophages increased the incorporation of [3H]-cholesterol and the cholesteryl ester content in smooth muscle cells, whereas unconditioned [3H]β-VLDL did not. On zonal ultracentrifugation of conditioned medium containing [3H]β-VLDL with macrophages, radioactivity was found at two peaks of density 1.150 and < 1.006. This new fraction with d = 1.150 (peak II) migrated at β-positions, the same as that of low-density lipoprotein (LDL) on agarose gel elec...