Gen Yoshino

Clinical Significance of Small Dense Low-Density Lipoprotein Cholesterol Levels Determined by the Simple Precipitation Method

Objective—Recently, we established a simple method for the quantification of small dense LDL cholesterol (C) using heparin-magnesium precipitation. The small dense LDL-C level was identical to cholesterol in the denser LDL fraction with a density of 1.044 to 1.063 g/mL. The aim of this study was to examine clinical significance of this precipitation method for small dense LDL-C. Methods and Results—Small dense LDL-C was measured by a direct homogenous LDL-C assay in the supernatant that remained after heparin-magnesium precipitation with density <1.044 lipoproteins. In 313 normolipidemic subjects, the mean value of small dense LDL-C was 31±13 mg/dL. In 462 healthy subjects, small dense LDL-C levels were positively correlated with serum triglyceride and LDL-C and were inversely correlated with high-density lipoprotein cholesterol (HDL-C). Combined hyperlipidemia showed the highest small dense LDL-C level among the various types of hyperlipidemia. Patients with type 2 diabetes had an increased small dense LDL-C level (55±17). Patients with coronary heart disease also had increased small dense LDL-C levels (53±30) irrespective of the presence of diabetes, whereas their LDL-C levels were comparable to those of normolipidemic controls (111±31 versus 104±22). Conclusion—These results suggest that measurement of small dense LDL-C by the present precipitation method is useful to evaluate atherogenic risk and may be applicable to routine clinical examination.

Increased cholesterol concentration in intermediate density lipoprotein fraction of normolipidemic non-insulin-dependent diabetics

There is increasing agreement about the atherogenicity of intermediate density lipoprotein (IDL). In order to determine whether normocholesterolemic diabetics are at a higher risk of atherosclerosis, cholesterol concentrations in three subclasses of triglyceride-rich lipoprotein fraction (Sf 12-400) were examined. Their plasma triglyceride and cholesterol levels were limited to below 150 and 250 mg/dl, respectively. They were divided into 3 groups according to their treatment: insulin injection (group I), sulphonylurea (group S) and diet alone (group D). Age-matched healthy normolipidemic non-obese subjects served as controls (group C). Triglyceride-rich lipoproteins were separated by ultracentrifugation: very low density lipoprotein (VLDL), Sf 60-400; intermediate density lipoprotein (IDL1), Sf 20-60; IDL2; Sf 12-20. Cholesterol concentrations in total plasma, VLDL, IDL2 and high density lipoprotein (HDL) were all identical in every group. A significant increase in cholesterol concentration was found in IDL1 of groups S and D. Low density lipoprotein-cholesterol of group I was also increased. These findings indicate an increased risk factor in normolipidemic diabetics.

Fasting insulin and leptin serum levels are associated with systolic blood pressure independent of percentage body fat and body mass index.

OBJECTIVE To examine the relationship between leptin and insulin serum levels and systolic and diastolic blood pressure in young men. SETTING Kobe University of Mercantile Marine, Kobe, Japan. PARTICIPANTS One hundred and ninety-eight male students aged 18-20 years (comprising 100% of those eligible). DESIGN AND MEASUREMENTS A cross-sectional survey of a sample of male college students was performed, with measurements to include anthropometry, blood pressure and blood tests after overnight fasting. RESULTS Compared with 90 men with an optimal blood pressure, 56 men with high-normal and high blood pressure had an increase in body mass index (23.7 +/- 5.2 versus 20.4 +/- 2.2 kg/m2), percentage body fat (21.7 +/- 8.0 versus 16.3 +/- 4.2%) and serum leptin (3.7 +/- 4.7 versus 1.5 +/- 0.8 ng/ml). In addition, they had greater serum insulin (59 +/- 31 versus 43 +/- 12 pmol/l) despite there being no differences in plasma glucose, resulting in a reduction of the ratio of glucose to insulin (x 10(6)) (107 +/- 43 versus 126 +/-, which is an estimate of insulin sensitivity in a nondiabetic population. Furthermore, the 56 men had higher serum triglyceride levels, although there was no difference in low density lipoprotein-cholesterol and high density lipoprotein-cholesterol between men with optimal and high-normal plus high blood pressure. Similar differences were found between men in a top versus low tertile of systolic and diastolic blood pressure. In multiple regression analysis, both log leptin and log insulin emerged as determinants for systolic blood pressure independent of body mass index and percentage body fat, but an association with diastolic blood pressure was only shown for log leptin. CONCLUSION Hyperleptinemia and hyperinsulinemia may be regulators of arterial pressure, independent of body mass index or percentage body fat.

Effect of CS-514 (pravastatin) on VLDL-triglyceride kinetics in rats

The effect of CS-514 (pravastatin; Sankyo Co., Tokyo), a competitive inhibitor of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, on triglyceride turnover, was studied in male Wistar rats. CS-514 (15 +/- 1 mg/day per rat) was administered as a 0.04% solution in drinking water for 14 days. Triglyceride and cholesterol in very low density lipoprotein (VLDL) and plasma triglyceride were reduced by treatment with CS-514. Plasma cholesterol level was not suppressed by CS-514. The CS-514 treated rats had a significantly suppressed triglyceride secretion rate (TgSR) during the fed state compared to control rats (0.85 +/- 0.1 vs. 1.07 +/- 0.3 mg/min, P less than 0.05). By contrast, CS-514 treatment did not suppress TgSR after an overnight fast. These data demonstrate that CS-514, an inhibitor of cholesterol biosynthesis can suppress VLDL-triglyceride secretion in rats and that this effect can be modified by dietary manipulation.

Long-term treatment of hypercholesterolemic non-insulin dependent diabetics (NIDDM) with pravastatin (CS-514)

We examined the long-term effect of pravastatin, a new potent inhibitor of endogenous cholesterol biosynthesis, on glucose and lipid metabolism in hyperlipidemic NIDDM. Ten patients (5 on sulfonylurea, 5 on diet) were studied over 12 months. Five were WHO type IIa and 5 were type IIb. Blood was taken before and then 1, 6 and 12 months after initiating 10 or 20 mg daily of pravastatin. The cholesterol concentration in whole plasma and very low density lipoprotein (VLDL), plasma triglyceride and apolipoprotein (apo) B were all significantly decreased within the first month. These changes lasted for 1 year. High density lipoprotein (HDL)-cholesterol increased in the first month but returned to base line thereafter. Low density lipoprotein (LDL)-cholesterol tended to decrease in the first month, and was suppressed significantly from the 6th month (11%) to the 12th month (16%). The effect of pravastatin on LDL-cholesterol in NIDDM was slower and weaker than that published for non-diabetic hypercholesterolemia. Therefore, the mechanism by which pravastatin suppresses plasma cholesterol levels in these two conditions may differ. After 1 year, no adverse effects were noted on hematopoietic, hepatic or renal function. Blood glucose level, hemoglobin A1c and the insulin response to oral glucose were unchanged. In addition, serum creatine phosphokinase showed no abnormal increase. Careful ophthalmological examinations before and after pravastatin treatment revealed no development of new lenticular opacities. Thus, pravastatin appears to be a safe and effective drug for the long-term treatment of NIDDM with hypercholesterolemia.

Abnormal Lipoprotein Composition in Normolipidemic Diabetic Patients

To see whether there are any lipoprotein abnormalities in diabetic patients without hyperlipidemia, lipoprotein composition was examined in 75 strictly normolipidemic diabetic patients. Their plasma cholesterol (chol) and triglyceride (TG) were limited to <6.0 and <1.7 mM, respectively. Body-weight- and age-adjusted normolipidemic healthy subjects served as the control group. Plasma total chol and TG and low-density lipoprotein (LDL-) and high-density lipoprotein (HDL-) chol were identical in the diabetic and control subjects. Total apolipoprotein B (apoB) in the plasma of the diabetic subjects was significantly elevated. The chol-apoB ratio in the TG-rich (very-low-density + intermediate-density) lipoprotein fraction (Sf 12-400) of the diabetic subjects was significantly higher than the control value, whereas LDL-apoB levels were increased and chol-apoB ratio in the LDL fraction was significantly suppressed in the diabetic subjects. Because each LDL particle contains only one apoB molecule, apoB and chol-apoB ratio in this fraction can represent particle number and chol loading of the LDL particles, respectively. Thus, these data suggest that LDL particle number is increased, and the particles are chol depleted in diabetic subjects even if they are normolipidemic.

VLDL Triglyceride Kinetics in Wistar Fatty Rats, An Animal Model of NIDDM: Effects of Dietary Fructose Alone or in Combination With Pioglitazone

The effects of dietary fructose alone or in combination with a new oral agent, pioglitazone, on VLDL-triglyceride (TG) turnover were studied in genetically obese Wistar fatty rats characterized by hyperinsulinemia (7,488 ± 954 pmol/l), hyperglycemia (22.5 ± 1.4 mmol/l), and hypertriglyceridemia (4.39 ± 0.54 mmol/l). They had an increased hepatic TG production (16.2 ± 0.1 μmol/min; lean rats, 5.4 ± 0.3 μmol/min) as well as a longer half-life of VLDL-TG from lean donors (8.8 ± 1.4 min, lean recipients; 2.3 ± 0.9 min). In addition, in lean recipients, the half-life of VLDL-TG from fatty donors was longer than that from lean donors (4.80 ± 0.56 vs. 3.14 ± 0.23 min). Although feeding fructose into fatty rats did not change plasma glucose and insulin levels, it produced a twofold increase in TG levels (8.74 ± 1.15 mmol/l). This was associated with a 1.7-fold increase in TG production to 27.5 ± 1.2 μmol/min, while no significant change was found in the half-life of lean VLDL-TG in fructose-fed fatty recipients (10.9 ± 2.4 min) or in that of VLDL-TG from fructose-fed fatty donors in lean recipients (4.46 ± 0.76 min). Daily administration of pioglitazone (3 mg/kg body weight) in fructose-fed fatty rats ameliorated glycemia and triglyceridemia to the level of lean rats (8.1 ± 0.7 and 1.18 ± 0.05 mmol/l, respectively) and insulinemia to a lesser extent (2,712 ± 78 pmol/l). A fall in TG levels was associated with improvement of an impairment in the ability of fructose-fed fatty rats to remove lean VLDL-TG (half-life: 2.6 ± 0.6 min). Pioglitazone, however, produced no change in TG production (25.9 ± 2.7 μmol/min), the half-life of VLDL-TG from fructose-fed fatty donors in lean recipients (4.17 ± 0.38 min), or the activity of lipoprotein lipase and hepatic lipase in postheparin plasma. We conclude that in Wistar fatty rats 1) hypertriglyceridemia is attributed to TG overproduction and impaired TG catabolism, and the latter is due to changes in both VLDL, such that they are less able to be removed, and changes in the nature of Wistar fatty rats, such that they are less able to remove VLDL-TG; 2) fructose further increases hepatic TG production with a resultant deterioration in hypertriglyceridemia; 3) pioglitazone normalizes TG levels by altering the physiology of the Wistar fatty rats in a manner that increases their ability to remove VLDL-TG from the circulation.

Effect of dietary fructose on triglyceride turnover in streptozotocin-diabetic rats

The effects of fructose or glucose on plasma triglyceride kinetics in streptozotocin (40 mg/kg) diabetic rats were studied using Triton WR1339. To separate groups of diabetic rats fructose or glucose was supplied at 10% in drinking water. Diabetic rats without sugar supplementation (diabetic control) had significantly suppressed triglyceride secretion compared to non-diabetic controls. Neither fructose nor glucose supplementation increased the triglyceride secretion rate in diabetic rats. However, despite reduced secretion rates, plasma triglyceride levels in glucose-supplemented diabetic rats, diabetic controls and non-diabetic controls were essentially identical. This suggested that removal of triglyceride from the circulation was impaired in the diabetic rats. In contrast, fructose supplementation resulted in a more than 150% (significant) increase in the mean plasma triglyceride of diabetic rats. The observation of significant hypertriglyceridemia in spite of low triglyceride secretion rate in fructose-supplemented diabetic rats suggests that dietary fructose, but not glucose, interferes with triglyceride removal from the circulation of streptozotocin-diabetic rats. This impairment by dietary fructose is in addition to the impaired triglyceride removal associated with diabetes alone.

Lecithin-cholesterol acyltransferase (LCAT) deficiency with a missense mutation in exon 6 of the LCAT gene.

The plasma enzyme, human lecithin-cholesterol acyltransferase (LCAT) is responsible for the majority of cholesterol ester formation in human plasma and is a key enzyme of the reverse transport of cholesterol from peripheral tissue to the liver. We sequenced genomic DNA of the LCAT gene from a Japanese male patient who was clinically and biochemically diagnosed as a familial LCAT deficiency. Analysis of all exons and exon-intron boundaries revealed only a single G to A transition within the sixth exon of both allele of the gene, leading to the substitution of methionine for isoleucinle at residue 293 of the mature enzyme. This mutation creates a new hexanucleotide recognition site for the restriction endonuclease Ndel. Familial study of Ndel digestion of the genomic DNA and determination of plasma LCAT activity established that the patient and his sister whose plasma LCAT activity were extremely reduced were homozygous and his children whose plasma LCAT activity were about half of normal controls were heterozygous for this mutation.

Somatostatin Concentration Responds to Arginine in Portal Plasma: Effects of Fasting, Streptozotocin Diabetes, and Insulin Administration in Diabetic Rats

Changes of somatostatin concentration in response to a single i.v. injection of arginine (400 mg/kg body weight) were examined in extracted portal plasma of normal and diabetic rats in the fully fed state and after 24 h of fasting, as well as in diabetic rats treated with insulin for one week. In both normal and diabetic animals fasted for 24 h, the basal level of somatostatin declined but the magnitude of the arginine-induced elevation of somatostatin was not affected, suggesting a physiologic role of the tetradecapeptide in nutrient homeostasis. When compared with intact rats, diabetic animals were shown to have increased levels of somatostatin before and after arginine administration, both of which were attenuated by insulin replacement therapy. These findings suggest that alterations of D cell function in streptozotocin diabetes may be related to either insulin deficiency or its metabolic consequences.