Seishi Nagano

Albuminuria is directly associated with increased plasma PAI-1 and factor VII levels in NIDDM patients

Increased plasma plasminogen activator inhibitor type 1 (PAI-1), coagulation factor VII (FVII) and fibrinogen levels have been recognized as risk factors for cardiovascular disease. Because a substantially high incidence of cardiovascular disease has been reported in diabetic patients with nephropathy compared with those without nephropathy, we measured plasma levels of PAI-1, FVII activity and fibrinogen in non-insulin-dependent diabetic patients (NIDDM) with normoalbuminuria (without nephropathy), microalbuminuria (incipient nephropathy) and macroalbuminuria (overt nephropathy). PAI-1 and FVII levels were significantly increased in NIDDM with overt nephropathy compared with NIDDM without nephropathy. Fibrinogen levels were comparable between the patients with normo-, micro- and macro-albuminuria. Univariate regression analysis indicated that PAI-1 and FVII levels were significantly correlated with the albumin excretion rate (AER) in urine. PAI-1, FVII and fibrinogen levels were significantly correlated with the degree of insulin resistance estimated by the steady state plasma glucose concentration (SSPG) during the continuous infusion of glucose, insulin and octreotide. PAI-1 levels were correlated with plasma triglyceride (TG) levels. Multiple regression analysis revealed that AER was significantly associated with PAI-1 and FVII levels, whereas TG lost significant correlation with PAI-1 when AER, SSPG and plasma TG were entered as independent variables. SSPG retained an independent correlation with fibrinogen, PAI-1 and FVII levels. These results suggest that elevated plasma levels of PAI-1 and FVII in NIDDM patients with nephropathy are directly associated with renal damage, whereas insulin resistance widely regulates hemostatic components in NIDDM patients, irrespective of the presence of nephropathy.

High prevalence of small LDL particles in non-insulin-dependent diabetic patients with nephropathy

To determine whether small-sized low density lipoprotein (LDL) is associated with a high incidence of coronary heart disease in diabetic nephropathy, we measured the LDL particle size in non-insulin-dependent diabetes mellitus (NIDDM) patients with various degrees of albuminuria (n = 95) and age-, weight-matched non-diabetic control subjects (n = 31). The diabetic subjects were divided into three groups, normoalbuminuric, microalbuminuric and macroalbuminuric NIDDM, based on the amount of albuminuria. The average diameter of LDL particles was determined by non-denaturing polyacrylamide gradient (2-16%) gel electrophoresis. The plasma lipid and lipoprotein concentrations were comparable between the non-diabetic controls and normoalbuminuric NIDDM, whereas the plasma triglyceride, very-low-density lipoprotein (VLDL) or LDL concentration was significantly increased in diabetic nephropathy. The mean LDL particle size was significantly smaller in microalbuminuric NIDDM compared with the controls or normoalbuminuric NIDDM, and the LDL size was further decreased in macroalbuminuric NIDDM. The incidence of small LDL (diameter < 255 A) was remarkably increased in microalbuminuric (58%) and macroalbuminuric NIDDM (67%) compared to the control (13%) and normoalbuminuric NIDDM (27%). Corresponding to the decreased LDL size, the cholesterol content of the LDL was significantly depleted in NIDDM with nephropathy. The high prevalence of small LDL in diabetic nephropathy was also observed even when hypertriglyceridemic or hypertensive subjects were excluded from each group. The increment in triglyceride-rich lipoprotein (d < 1.006) after oral fat-loading was increased, and postheparin lipoprotein lipase activity was decreased significantly in diabetic nephropathy. These abnormalities were significantly associated with LDL particle size. Multivariate regression analysis revealed that the amount of albuminuria was closely associated with the average LDL particle size, and this association was independent of the plasma triglyceride level. Neither insulin resistance nor glycemic control was directly associated with LDL particle diameter. The present study indicates that LDL particles become smaller in diabetic nephropathy, and this may be associated primarily with abnormal triglyceride metabolism. However, in addition to hypertriglyceridemia, other metabolic abnormalities caused by diabetic nephropathy may also be involved in the pathogenesis of small LDL particles.

Effect of Pravastatin sodium, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on very-low-density lipoprotein composition and kinetics in hyperlipidemia associated with experimental nephrosis

The effect of Pravastatin sodium (CS-514), a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA R) on very-low-density lipoprotein (VLDL) composition and kinetics was studied in normal and experimental nephrotic rats under fasting conditions. Nephrotic rats, induced by a single intraperitoneal injection of puromycin aminonucleoside (100 mg/kg body weight), had significantly higher plasma lipids and apoprotein (apo) B concentrations than controls. The hypertriglyceridemia associated with nephrosis was mainly due to a markedly elevated VLDL-triglyceride (TG) concentration. Pravastatin sodium was administrated as a 0.04% solution in drinking water for 7 days to normal control and nephrotic rats. Plasma TG concentration in both control and nephrotic rats was significantly reduced by the treatment with Pravastatin, but plasma cholesterol levels were not reduced by the treatment in either group of rats. TG, cholesterol, phospholipid, and apo B concentrations in nephrotic VLDL were significantly reduced by Pravastatin treatment, whereas only TG was decreased in control VLDL. Pravastatin reduced the apo B 100 + 95/48 ratio in nephrotic VLDL. Pravastatin did not alter the lipid concentration of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in control and nephrotic rats. VLDL-TG turnover studies showed that TG secretion rate was significantly suppressed by Pravastatin administration without affecting its removal in both groups of rats. These suggested that Pravastatin, an inhibitor of cholesterol biosynthesis, can reduce VLDL concentration by rectifying the overproduction of VLDL exhibited in nephrotic rats.

The Lowering Effect of Probucol on Plasma Lipoprotein and Proteinuria in Puromycin Aminonucleoside-Induced Nephrotic Rats

Hyperlipidemia associated with nephrotic syndrome was treated with probucol and the changes in plasma lipoprotein lipid concentration and urinary protein excretion were examined in puromycin aminonucleoside-induced nephrotic rats. Rats made nephrotic exhibited severe hyperlipidemia with increases in all major lipoprotein fractions. Probucol treatment of nephrotic rats significantly lowered plasma triglyceride (TG), cholesterol (Ch) phospholipid (PL) and apoprotein B associated with very-low-density and low-density lipoprotein and Ch and PL in high-density lipoprotein (HDL). Malondialdehyde (MDA) associated with the lipoproteins was significantly elevated in nephrotic rats and probucol treatment also lowered MDA concentration in all major lipoproteins. In control rats probucol moderately, but significantly, reduced plasma TG and HDL-Ch concentrations. Proteinuria associated with nephrosis was decreased significantly by treatment with probucol. Probucol treatment did not affect blood urea nitrogen and plasma creatinine levels. A significant positive correlation existed between the amount of protein excreted in urine and the plasma lipid concentrations in all nephrotic rats, suggesting that the hypolipidemic effect of probucol may attenuate proteinuria associated with nephrosis. These results suggest that probucol may be a favorable treatment for hyperlipidemia associated with nephrotic syndrome.

Correlation of insulin deficiency and hypertriglyceridemia in diabetic rats

The relationship between plasma insulin (IRI) and lipid concentration, or triglyceride (TG) kinetics was studied in streptozotocin-induced diabetic rats (DM) to examine how insulin deficiency is associated with the mechanism of hypertriglyceridemia (HTG) in diabetes. Plasma glucose and ketones were significantly elevated and IRI reduced in DM. Plasma glucagon concentration in DM was similar to controls. The plasma concentration of TG, total cholesterol phospholipid and apoprotein B was 3-4-fold higher in DM compared to control rats. The HTG in DM was mainly attributable to an increase in the concentration of TG-rich lipoprotein (TRL). Multiple linear regression analysis showed a positive relationship between the concentration of non-esterified fatty acid and plasma lipids, but the decrease in IRI best correlated with increased concentrations of lipids and apoprotein B in plasma and TRL. Neither glucose nor glucagon correlated significantly with lipids or apoprotein B concentration in plasma or TRL. The rate of entry of TG into blood was similar between DM and controls, and in DM this significantly correlated with IRI. Clearance of radiolabeled TRL-TG in DM was significantly decreased and correlated with IRI. Conversely, the removal of radiolabeled Intralipid-TG was similar for DM and controls. The data suggest that insulin critically regulates TRL-TG metabolism in DM and that a catabolic defect of TRL-TG due to insulin deficiency is a main reason for the HTG.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of hypertriglyceridemia in dahl salt-sensitive rats, an animal model of spontaneous nephrotic syndrome

It has been reported that focal and segmental glomerulosclerosis (FSGS) with pronounced proteinuria rapidly develop in Dahl salt-sensitive hypertensive (DS) rats fed a high-salt diet. We found that even when they are fed a standard rat chow (0.3% NaCl), DS rats, especially males, exhibit marked proteinuria, hypoalbuminemia, and hypertriglyceridemia without marked hypertension at 32 to 38 weeks of age. The nephrosis was associated with spontaneously developed FSGS. We therefore investigated the mechanism of hypertriglyceridemia in nephrotic animals. Plasma triglyceride (TG) and apoprotein (apo) B levels were markedly increased in DS rats compared with Sprague-Dawley (SD) rats, and this was mainly attributable to an increase in the concentration of very-low-density lipoprotein (VLDL). The TG secretion rate estimated by the Triton WR1339 method was significantly greater in DS rats. VLDL-TGs isolated from both the DS and SD rats were endogenously radiolabeled with different isotopes, and a mixture of these was then injected into DS and SD recipients. The half-life of VLDL-TG was about three times longer in DS recipients, regardless of the source of VLDL. In SD recipients, VLDL from DS rats was cleared at a slower rate than VLDL from SD rats. The activity of lipoprotein lipase in postheparin plasma was substantially decreased in DS rats. Isoelectric focusing gel electrophoresis (IEF) showed that the ratio of apo E/C or apo C-II/C-III in VLDL was markedly decreased and the ratio of apo E or apo C to apo A1 in high-density lipoprotein (HDL) was slightly decreased in DS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of acute hyperglycemia on plasma triglyceride concentration and triglyceride secretion rate in non-fasted rats.

The effect of an intravenous infusion of glucose on plasma triglyceride (TG) concentration in fed rats was determined in order to partially elucidate the mechanism of diabetes-induced hypertriglyceridemia. Glucose infused at 8 mg/kg per min caused the plasma TG concentration to be elevated significantly when compared to controls infused with saline alone. In rats which were euglycemic (clamped, insulin infused at 2.5 mU/kg per min), plasma TG concentration remained constant throughout the glucose infusion period (8 mg/kg per min). Hyperglycemic rats infused with insulin (2.5 mU/kg per min) as well as with glucose (16 mg/kg per min) were also hypertriglyceridemic. Infusion of insulin alone did not change the concentration of plasma TG over a 150 min period. Glucose was also infused (8 mg/kg per min) with somatostatin (1 micrograms/kg per min) to block endogenous production of insulin. Somatostatin infusion did not suppress glucose-induced hypertriglyceridemia. For all treatments, the net change in TG concentration was found to positively correlate with the net change in plasma glucose concentration at 150 min after the infusions (r = 0.83, P less than 0.001). The higher TG concentration in the glucose infused, hyperglycemic clamp and glucose plus somatostatin groups reflected an increased rate of TG secretion, in the presence of a lower concentration of plasma free fatty acids. These results suggest that in a non-fasted state, acute hyperglycemia increases plasma TG by stimulating hepatic TG secretion, in a manner which is independent of either plasma insulin or free fatty acids levels.

Similar to oleic acid, eicosapentaenoic acid stimulates apolipoprotein B secretion by inhibiting its intracellular degradation in Hep G2 cells

We previously reported that oleic acid (OA) rapidly increased apolipoprotein (apo) B secretion by suppressing early intracellular degradation of nascent apo B in Hep G2 cells and suggested that the suppression of apo B degradation is associated with triglyceride (TG) biosynthesis from OA. To determine whether the inhibition of apo B degradation is associated with increased TG synthesis or is a direct effect of OA, we examined the effect of another fatty acid, eicosapentoenoic acid (EPA), on apo B kinetics in Hep G2 cells, since it is well known to have hypolipidemic action in clinical studies. The incorporation of [3H]glycerol into cellular TG was stimulated five-fold when Hep G2 cells were incubated for 2 h with EPA or OA (0.4 or 0.8 mM-1.5% bovine serum albumin (BSA) complex). The incorporation of [14C]acetic acid into cellular cholesteryl ester (CE) was significantly decreased by EPA treatment, whereas OA did not affect CE synthesis. Similar effects of these fatty acids on cellular lipid synthesis were observed in long-term incubation (24 h). Apo B was linearly secreted into the medium during 3 h, and EPA and OA doubled the rate of secretion. In long-term (24 h) incubations, both fatty acids significantly increased the incorporation of [3H]leucine into secreted apo B radioactivity or the accumulation of apo B mass in the medium. Pulse-chase studies revealed that both EPA and OA reduced intracellular apo B degradation to a similar degree. The inhibition of apo B degradation was also observed when the cells were preincubated with either EPA or OA for 24 h. These results suggest that increased TG synthesis leads to suppression of intracellular apo B degradation, which is independent of the source of exogenous fatty acid.

Fluvastatin, a New Inhibitor of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, Suppresses very Low-Density Lipoprotein Secretion in Puromycin Aminonucleoside-Nephrotic Rats

The effects of fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the hyperlipidemia associated with nephrosis were studied. Nephrotic rats, induced by a single intraperitoneal injection of puromycin aminonucleoside (100 mg/kg body weight), had significantly higher plasma triglyceride (TG), total cholesterol and apoprotein (apo) B concentrations than controls. Fluvastatin was administrated as a 0.01% solution in drinking water for 14 days to either normal control or nephrotic rats. Concentrations of TG and apo B in plasma, and very low-density lipoprotein (VLDL) in nephrosis were completely normalized by the treatment with fluvastatin, but concentrations of cholesterol in plasma and each lipoprotein fraction were not altered by the treatment. The ratio of apo E to C in VLDL was significantly decreased in nephrotic rats, but the fluvastatin treatment increased this ratio. TG secretion rate estimated by the Triton WR1339 method was significantly increased in nephrotic rats, but was normalized by fluvastatin. Percent composition of TG in newly secreted VLDL particles in post-Triton plasma was not decreased by fluvastatin treatment, suggesting that the number of newly secreted VLDL particles was reduced by the treatment. Postheparin plasma lipolytic activities were not affected by the fluvastatin treatment. These results demonstrate that fluvastatin can effectively ameliorate the high concentration of VLDL by suppressing the hepatic secretion in nephrotic rats, and suggest that an inhibition of cholesterol biosynthesis suppresses VLDL secretion from the liver.

Involvement of Lipoproteins in Suppression of Interleukin 2-Dependent Cell Proliferation by Sera from Aged Humans

Sera from the elderly, but not young, inhibited markedly interleukin 2 (IL-2)-dependent proliferation of cloned T cells. Sera of aged humans contained high levels of triglyceride, cholesterol, very low density lipoproteins (VLDL) and low density lipoproteins (LDL). VLDL and LDL inhibited the proliferation in a dose-related manner. The inhibition correlated with levels of serum VLDL and LDL. These results suggest that lipoproteins may act as a regulator of IL-2-dependent proliferation and that disturbance of lipid and lipoprotein metabolism may be partially responsible for diminished immune responses in the elderly.