Decreased release of lipoprotein lipase is associated with vascular endothelial damage in NIDDM patients with microalbuminuria.

OBJECTIVE: To explore mechanisms for hypertriglyceridemia in diabetic patients with microalbuminuria, we examined an association between heparin-releasable lipoprotein lipase (LPL) and the von Willebrand factor (vWF), based on the hypothesis that LPL bound to endothelium is decreased by generalized endothelial damage. RESEARCH DESIGN AND METHODS: A total of 37 NIDDM patients with microalbuminuria and 69 patients with normoalbuminuria were studied. Plasma LPL mass in post-heparin plasma and plasma vWF antigen were quantified by sandwich-enzyme immunoassay and enzyme-linked immunosorbent assay, respectively. RESULTS: The NIDDM patients with microalbuminuria had higher plasma triglyceride (TG) and lower HDL cholesterol concentrations compared with the patients with normoalbuminuria. Heparin-releasable LPL mass was significantly lower in the microalbuminuric than in the normoalbuminuric subjects. Plasma level of vWF, a marker for endothelial damage, was significantly increased in microalbuminuric subjects compared with their normoalbuminuric counterparts. The LPL mass was inversely correlated with plasma vWF level at a high correlation coefficient value. The LPL mass was inversely related to TG and positively to HDL cholesterol concentrations. CONCLUSIONS: These results suggest that widespread endothelial damage occurred in NIDDM patients with microalbuminuria, thereby LPL moiety bound to the endothelium is decreased, which results in an impaired catabolism of TG-rich lipoproteins.

High prevalence of small dense LDL in diabetic nephropathy is not directly associated with kidney damage: a possible role of postprandial lipemia.

To determine whether high prevalence of small dense low-density lipoprotein (LDL) in non-insulin-dependent diabetes (NIDDM) with nephropathy is directly associated with kidney damage, we measured LDL particle size by non-denaturing 2-16% gradient polyacrylamide gel electrophoresis in non-diabetic patients with primary renal disease and compared the results to particle size in NIDDM patients with diabetic nephropathy. The average LDL particle diameter was significantly smaller in patients with diabetic nephropathy (245+/-3 A mean +/- SEM) compared to the controls (263+/-1 A), diabetics without nephropathy (257+/-2 A), patients with primary renal disease (254+/-2 A) or non-diabetic patients treated with hemodialysis (HD) (260+/-1 A). The incidence of small LDL (mean diameter is < or =255 A) was remarkably increased in diabetic nephropathy (67%) compared to diabetes without nephropathy (27%), patients with renal disease (24%), HD patients (15%) and controls (10%). LDL size in patients with primary renal disease was significantly smaller than those in controls. However, because there was an excellent correlation between LDL size and plasma triglyceride (TG) levels, when hypertriglyceridemic subjects (TG >1.7 mM) were excluded, no difference of LDL size was observed between the renal disease group (260+/-2 A) and the control group (264+/-1 A). On the other hand, even when hypertriglyceridemic subjects were excluded, LDL size was still smaller in diabetic nephropathy (250+/-4 A). We performed an oral fat load test in normotriglyceridemic subjects (fasting TG <1.7 mM) of control, diabetes with and without nephropathy and primary renal disease. The TG responses in plasma and TG-rich-lipoprotein (TRL) (d <1.006) after the oral fat load were significantly greater in NIDDMs with nephropathy compared to controls or NIDDMs without nephropathy, while such a marked postprandial lipemia was not observed in patients with primary renal disease. In these fasting normotriglyceridemic subjects, LDL size was significantly inversely correlated with postprandial TG responses, which is totally independent from fasting TG levels. These results suggest that high prevalence of small dense LDL in NIDDM patients with nephropathy is not directly associated with kidney damage. Postprandial lipemia may play an important role in reducing LDL particle size in these patients.

Doxazosin reduces prevalence of small dense low density lipoprotein and remnant-like particle cholesterol levels in nondiabetic and diabetic hypertensive patients.

Small dense low density lipoprotein (LDL) and remnant lipoproteins are potent atherogenic lipoproteins, often elevated in the plasma of patients with type 2 diabetes. The alpha1-blocker doxazosin has been reported to favorably affect the plasma lipid profile. We examined whether doxazosin could reduce these atherogenic lipoproteins in hypertensive subjects with and those without type 2 diabetes. Seventeen nondiabetic hypertensive patients and 33 hypertensive patients with type 2 diabetes were studied. Doxazosin (2 to 4 mg) was administered alone or with other previously received antihypertensive drugs for 6 months. Mean LDL size was measured by 2% approximately 16% gradient gel electrophoresis. Remnant-like particle (RLP)-cholesterol was measured with the use of an affinity column containing anti-apoA1 and B100 monoclonal antibodies. Doxazosin effectively decreased blood pressure (BP) without significantly affecting glucose, glycosylated hemoglobin (HbA1c), or C-peptide levels in both nondiabetic and diabetic patients. Doxazosin significantly reduced triglyceride, apo CIII, and apo B, but did not alter total-, LDL- or HDL-cholesterol. Mean LDL particle diameter was significantly increased from 25.6+/-0.6 nm to 25.9+/-0.4 nm (P < .001) by doxazosin treatment, regardless of the presence of diabetes. Consequently, the prevalence of small dense LDL (<25.5 nm) was halved in both groups. The increase in LDL size significantly correlated with decrease in triglyceride level (r=-0.798, P < .0001). Doxazosin significantly reduced RLP-cholesterol in both groups. These results suggest that doxazosin may help to prevent coronary artery disease by reducing atherogenic lipoproteins, including small dense LDL and remnant lipoproteins, in hypertensive patients, regardless of the presence of diabetes.

Vascular endothelial markers, von Willebrand factor and thrombomodulin index, are specifically elevated in type 2 diabetic patients with nephropathy: comparison of primary renal disease.

To elucidate the hypothesis that albuminuria in diabetic subjects reflects widespread vascular damage, plasma markers for vascular endothelial damage was measured in diabetic subjects with various degrees of albuminuria and compared to results in patients with primary renal disease. The groups consisted of 31 non-diabetic patient controls with normoalbuminuria, 109 type 2 diabetic patients with normo- micro- and macro-albuminuria, and 16 proteinuric patients with primary renal disease. Endothelial markers, plasma von Willebrand factor (vWF) and thrombomodulin (TM), were measured by enzyme-linked immunosolvent assay and enzyme immunoassay (EIA) methods, respectively. Plasma vWF levels were similar in controls (119+/-7%, mean+/-S.E.M.) and diabetic patients with normoalbuminuria (139+/-6), but significantly elevated in diabetic patients with microalbuminuria (174+/-11) and macroalbuminuria (204+/-17), while the level was not increased in patients with primary renal disease (124+/-11). Because plasma TM level was strongly affected by kidney function, TM index (TM (FU/ml)/serum creatinine (mg %)) was used as an endothelial marker. The TM index was substantially increased in diabetic patients with overt nephropathy compared with controls (5.29+/-2.98 vs. 2.35+/-0.85), whereas this was not observed in patients with primary renal disease (3.25+/-0.29). Both vWF and TM index were significantly higher in diabetic patients with retinopathy than in the patients without retinopathy. These results suggest that generalized vascular endothelial damage occurs in diabetic nephropathy including the microalbuminuric stage, which is not attributed to kidney damage per se.

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.

Increase in mitochondrial GOT (m-GOT) activity after chronic alcohol consumption: clinical and experimental observations.

In order to clarify the origin and the mechanism of increased serum activity of glutamic oxalacetic transaminase (GOT) in chronic alcoholics, clinical and experimental investigations were carried out. Mitochondrial (m-GOT) and cytosolic GOT (c-GOT) isoenzymes were separated chromatographically by using a mini-column packed with Sephadex A50. Sixty percent of 63 alcoholics had elevated serum GOT. The m-GOT activity in alcoholics with total serum GOT activity of over 50 Karmen Units was 17.2 +/- 1.6 K.U. and the m-GOT/GOT ratio was the highest when compared to those in non-alcoholic liver diseases. In in vitro study, six hours of incubation of isolated hepatocytes from rats fed ethanol chronically resulted in an increased leakage of m-GOT into the incubation medium and also showed a tendency of a higher m-GOT/GOT ratio than that from control rats. The m-GOT activity thus released into the medium showed a highly significant inverse correlation with the viability of hepatocytes. These data suggest that m-GOT substantially contributes to an increased serum GOT often observed in chronic alcoholics.

Ten year survival rate of 131 patients with liver cirrhosis excluded the association of liver carcinoma at the establishment of diagnosis.

The present study reported 10 year suvival rate of 131 patients with liver cirrhosis excluded the association of liver carcinoma. Survival rate was calculated according to the life table method of Cutler and Ederer. One hundred thirty-one patients were diagnosed between 1965 and 1972. The overall 5 and 10 year survival rates of 131 patients were 58.3 +/- 4.7% and 36.5 +/- 6.2%, respectively. The 5 and 10 year survival rates of 56 patients with decompensated liver cirrhosis were 32.9 +/- 6.9% and 19.0 +/- 6.9%. The 5 year survival rate of 28 patients with prominent ascites was 20.6 +/- 8.4% and that of 14 patients with the rupture of esophageal varices was 29.4 +/- 13.4%. The improved treatment of decompensated liver cirrhosis and the exclusion of the patients with the association of liver carcinoma might contribute to these improved survival rates.

A case of subacute hepatitis with reversible liver fibrosis.

Reversibility of hepatic fibrosis was demonstrated histologically in a case with subacute hepatitis. His first liver biopsy showed the transformation to liver cirrhosis. Gamma-globulin, TTT and ZTT gradually recovered to normal range, and serial second to fourth liver biopsy revealed the gradual decrease in newly formed fibrous tissues of the liver.

Stimulation of interstitial collagenase in co-cultures of rat hepatocytes and sinusoidal cells.

Although the fibrosis observed during chronic liver injury is the result of a complex process, the striking accumulation of collagen in end stage liver disease has provoked interest in the mechanisms that regulate both collagen production and degradation in the diseased liver. The present studies have examined the cell interactions that may be important in the regulation of collagen degradation. Although minimal amounts of interstitial collagenase activity were noted in cultures of normal hepatocytes and sinusoidal cells, the co-cultures of these cells in the presence of lipopolysaccharide showed a substantial increase in collagenase activity. When the hepatocytes were obtained from rats that had been treated with carbon tetrachloride in vivo, the enhanced activity seen in the co-cultures did not require the addition of lipopolysaccharide. Further characterization of this interaction suggested that the increase in collagenolytic activity was partially due to the elaboration of soluble factors by the hepatocyte, which stimulated collagenase production by the sinusoidal cell population. Elaboration of collagenase activity by the sinusoidal cells was inhibited by cycloheximide, suggesting that protein synthesis was required. The proteolytic activity was abrogated by inhibitors of metalloproteinases but not by serine or thiol proteinase inhibitors. The degradation products of type I collagen were typical of the expected products seen with vertebrate collagenases. Thus, it appears that the increased collagenolytic activity detected in this co-culture system is attributable to the production of interstitial collagenase by the sinusoidal cell population. Such cell-cell interactions may play an important role in the maintenance of normal connective tissue structure of the liver during disease processes.

Collagenase production by rabbit liver cells in monolayer culture.

The mechanism of collagenase production by cells derived from rabbit liver was investigated in the present study. Fibroblasts alone, second to fourth passage cells, did not produce collagenase even if PMA, a potent inducer of collagenase production, was added to the culture medium. Collagenase activity was detected only in first passage cells, containing hepatocyte-like and fibroblast-like cells, after the addition of PMA. During 9 days of culture, there was negligible collagenase activity in the first 3 days, but in the following 3 days the activity was 2.54 +/- 0.31 U (mean +/- S.E.M., n = 5) (micrograms of collagen degraded per minute) per milligram of cell protein, and in the final 3 days the activity was 9.05 +/- 0.38 U/mg of cell protein. These results suggest that interaction between mesenchymal and parenchymal cells is important for collagenase production in the liver.

Different appearance of hepatic collagenase and lysosomal enzymes in recovery of experimental hepatic fibrosis.

1. Both activities of hepatic collagenase and lysosomal enzymes (acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase) have been observed in the recovery from experimental hepatic fibrosis in rats treated with carbon tetrachloride for 6 to 20 weeks, and compared with the disappearance of newly formed collagen fibers in the recovery process. 2. In the process of experimental hepatic fibrosis, collagenase activity reached maximum on sethe accumulation of collagen fibers in reversible hepatic fibrosis, but decreased to the same level as that of non-treated rat liver in cirrhotic stage. In the reocvery from reversible hepatic fibrosis, collagenase activity reached maximum on second day after the discontinuation of carbon tetrachloride, and decreased to the same extent of that of non-treated rat liver on seventh day. 3. Lysosomal enzyme activity was parallel to the activity of hepatic collagenase and to the accumulation of collagen fibers in the process of hepatic fibrosis. In the recovery stage, lysosomal enzyme activity in mesenchymal cells within the septa increased markedly on second day after the discontinuation of toxic agent but turned to the same level of that of non-treated rat liver seven days later, which was consistent with the appearance and disappearance of collagenase activity. On the other hand the appearance of lysosomal enzymes activities in Kupffer cells and hepatocytes was different from that of collagenase activity. That is lysosomal enzyme activity in Kupffer cells decreased in early days but increased five days later, and the enzyme activity in hepatocytes markedly decreased but gradually recovered to normal level seven days later. 4. The appearance of collagenase was observed at the beginning of the recovery stage. It indicates that mammalian collagenase initiates the collagen degradation and lysosomal enzymes might have a role in the subsequent degradation of collagen.

Studies on the metabolism and disposition of the new retinoid 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid. 5th communication: factors affecting percutaneous absorption in rats.

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl] benzoic acid (CAS 94497-51-5, Am-80) is a new synthetic retinoid which has been shown to have a potent topical antipsoriatic activity. Factors affecting the percutaneous absorption of Am-80 were studied with the intention of obtaining information for toxicity and clinical investigations. The percutaneous absorption of radioactivity was compared after topical application of 0.1% 14C-Am-80 ointment to female rats by simple application (SA), occlusive dressing technique (ODT), and application using lint (AUL). After single topical application to normal skin female rats, the percutaneous absorption of radioactivity was very low with no significant differences in the extent of absorption among the three application methods. In the stripped skin female rats, a distinct increase of the percutaneous absorption was observed indicating that it was markedly affected by the lack of the stratum corneum. Compared with the single dosing, a considerable increase of percutaneous absorption was observed following repetitive topical application once daily for 4 or 7 days to the normal skin female rats. The extent of increase was highest in ODT rats followed by SA rats, but was relatively low in AUL rats. The effects of concentration, dose and application area of 14C-Am-80 ointment on the percutaneous absorption of radioactivity were studied following topical application of 0.002%-0.008% 14C-Am-80 ointment to normal skin male rats by ODT to areas of 72 cm2/kg-360 cm2/kg (5%-25% of the body surface area) at ointment doses of 2g/kg-8 g/kg. When the application area and the amount of ointment applied were fixed at 144 cm2/kg (10% of the body surface area) and 2 g/kg, respectively, the amount of radioactivity absorbed increased in proportion to the 14C-Am-80 concentration in the ointment, whereas the rate of percutaneous absorption, expressed as the percent of dose, was nearly constant. When the 14C-Am-80 concentration in the ointment and the amount of ointment applied were fixed at 0.08% and 2 g/kg, respectively, both the amount of radioactivity absorbed and the rate of percutaneous absorption markedly increased with an increase in the application area. When the concentration of 14C-Am-80 in the ointment was set at 0.008% and the application area at 72 cm2/kg, 144 cm2/kg or 288 cm2/kg (5%, 10% or 20% of the body surface area), the amount of radioactivity absorbed increased as the amount of ointment applied increased for areas of the same, though the rate of percutaneous absorption remained almost constant. When the 14C-Am-80 concentration in the ointment was fixed at 0.008%, the amount of radioactivity absorbed increased markedly about 20-fold with 4-fold simultaneous increases in both the application area (from 72 cm2/kg to 288 cm2/kg) and the amount of ointment applied (from 2 g/kg to 8 g/kg).