Apolipoprotein changes associated with the plasma lipid-regulating activity of gemfibrozil in cholesterol-fed rats

Gemfibrozil (Lopid) is a new plasma lipid-regulating drug that decreases very low and low density lipoprotein (VLD/LDL) and increases high density lipoprotein (HDL) concentrations in man. The present experiments tested the effects of gemfibrozil on plasma lipoproteins and apolipoproteins in rats fed high fat/high cholesterol diets. Compared to chow-fed rats, cholesterol feeding for 2 weeks (20% olive oil/2% cholesterol) produced the expected increases in VLDL and intermediate density lipoprotein (IDL) while lowering plasma HDL. This was documented by using three methods of lipoprotein isolation: sequential ultracentrifugation, density gradient ultracentrifugation, and agarose gel filtration. Gemfibrozil gavaged at 50 mg/kg per day for 2 weeks during cholesterol feeding prevented these changes such that lipoprotein patterns were similar to those in chow-fed animals. Whole plasma apoE and apoA-I concentrations were decreased and apoB increased due to cholesterol feeding as determined by electroimmunoassay, but again gemfibrozil treatment prevented these diet-induced alterations. Gradient polyacrylamide gel electrophoresis patterns of the total d less than 1.21 g/ml lipoprotein fractions reflected the changes in apolipoprotein concentrations and further demonstrated a greater increase of apoBl compared to apoBh in cholesterol-fed rats. Gemfibrozil lowered the concentration of both apoB variants and prevented the shift of apoE from HDL to lower density lipoproteins. Changes in the distribution of apoE were confirmed using agarose gel column chromatography followed by electroimmunoassay. These methods also revealed a shift of apoA-IV from HDL to the d greater than 1.21 g/ml, lipoprotein-free fraction with gemfibrozil treatment when blood was taken from fasted or postabsorptive animals. Since it was also noted that in chow-fed rats more apoA-IV was present in the d greater than 1.21 g/ml fraction in the postabsorptive or fed state compared to fasted animals, it could be postulated that the shift of apoA-IV into this fraction in gemfibrozil-treated rats is related to an accelerated clearance of chylomicrons. It is concluded that gemfibrozil largely prevents the accumulation of abnormal lipoproteins in this model of dyslipoproteinemia, and that apoE may play a critical role in this normalization process.     

Metabolism of two forms of apolipoprotein B of VLDL by rat liver

Apolipoprotein B (apoB) is composed of metabolically distinct fractions of higher molecular weight (apoBh) and lower molecular weight (apoBl). When 125I-labeled very low density lipoprotein (VLDL) prepared from recirculating liver perfusates was injected into rats, labeled apoBl was preferentially removed from the plasma and apoBh entered low density lipoprotein (LDL). The time-related movement of labeled apoBh into higher density fractions was independent of that of labeled apoBl. When 125I-labeled triglyceride-rich lipoprotein (TRL) prepared from sucrose-fed rats was incubated with plasma from rats injected with heparin and then studied in a recirculating liver perfusion, apoBl was preferentially removed compared to apoBh. Thus, the loss of apoBl of hepatic VLDL in vivo was similar to the loss of apoBl of lipase-treated TRL in vitro. In control perfusions where TRL was incubated with heat-treated postheparin plasma, not only was there less initial hepatic clearance of apoB but the early phase of preferential apoBl removal during 30 min of perfusion was not observed. ApoE removal from perfusates was the same whether or not the TRL had been treated with heparin-releasable lipases. Apoprotein degradation, as indicated by the appearance in the perfusate of labeled degradation products, occurred 30 min after the preferential phase of apoBl removal. These results suggest that hepatic clearance of VLDL and TRL remnants is favored by lipolysis and by the presence of apoBl on the particle that enhances their hepatic binding and degradation.     

Apolipoprotein B overproduction by the perfused liver of the St. Thomas' mixed hyperlipidemic (SMHL) rabbit

The St. Thomas' mixed hyperlipidemic (SMHL) rabbit (previously St. Thomas' Hospital rabbit) is a putative model of familial combined hyperlipidemia (FCH). When fed a low (0.08%) cholesterol diet, it exhibits elevations in both plasma cholesterol and triglyceride compared to New Zealand White (NZW) controls. To determine the mechanism for this hyperlipidemia we studied the secretion of apolipoprotein B (apoB)-containing lipoproteins from perfused livers of both young and mature rabbits. During a 3-h perfusion we measured the total cholesterol and triglyceride content of the medium and the cholesterol, triglyceride, and apoB content of very low density lipoprotein (VLDL)(1) (S(f) 60;-400), VLDL(2) (S(f) 20;-60), intermediate (S(f) 12;-20), and low (S(f) 0;-12) density lipoproteins (IDL, LDL). Lipoprotein concentrations increased linearly throughout the perfusion period. The rate of cholesterol output was 3-fold higher (459 vs. 137 ng/g liver/min, P = 0.003) in SMHL versus NZW rabbits whilst that of triglyceride was similar (841 vs. 662 ng/g liver/min, NS). VLDL(1) cholesterol output was elevated 2-fold (232 vs. 123 ng/g liver/min, P < 0.05) and VLDL(2) + IDL + LDL cholesterol output, 4.5-fold (106 vs. 23 ng/g liver/min, P < 0. 005) in SMHL versus NZW rabbits. ApoB output in VLDL1 was 38 ng/g liver per min in SMHL and 14 ng/g liver per min in NZW (NS). In SMHL VLDL(2) + IDL + LDL apoB was increased 9-fold at 53 versus 6 ng/g liver per min in NZW (P < 0.001). We conclude that the SMHL rabbit overproduces apoB-containing lipoproteins particularly in the VLDL(2) + IDL + LDL fraction, a characteristic consistent with its use as a model of FCH.     

Effect of apolipoprotein E genotype on apolipoprotein B-100 metabolism in normolipidemic and hyperlipidemic subjects

The effect of apolipoprotein (apo) E genotype on apoB-100 metabolism was examined in three normolipidemic apoE2/E2, five type III hyperlipidemic apoE2/E2, and five hyperlipidemic apoE3/E2 subjects using simultaneous administration of ¹³¹I-VLDL and ¹²⁵I-LDL, and multi-compartmental modeling. Compared with normolipidemic apoE2/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased plasma and VLDL cholesterol, plasma and VLDL triglycerides, and VLDL and intermediate density lipoprotein (IDL) apoB concentrations (P < 0.05). These abnormalities were chiefly a consequence of decreased VLDL and IDL apoB fractional catabolic rate (FCR). Compared with hyperlipidemic E3/E2 subjects, type III hyperlipidemic E2/E2 subjects had increased IDL apoB concentration and decreased conversion of IDL to LDL particles (P < 0.05). In a pooled analysis, VLDL cholesterol was positively associated with VLDL and IDL apoB concentrations and the proportion of VLDL apoB in the slowly turning over VLDL pool, and was negatively associated with VLDL apoB FCR after adjusting for subject group. VLDL triglyceride was positively associated with VLDL apoB concentration and VLDL and IDL apoB production rates after adjusting for subject group. A defective apoE contributes to altered lipoprotein metabolism but is not sufficient to cause overt hyperlipidemia. Additional genetic mutations and environmental factors, including insulin resistance and obesity, may contribute to the development of type III hyperlipidemia.     

Metabolism of apoprotein B in selectively bred baboons with low and high levels of low density lipoproteins

Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) apoprotein (apo)-B turnover rates were measured simultaneously by injecting 131I-labeled VLDL and 125I-labeled LDL into fasting baboons (Papio sp.) selectively bred for high serum cholesterol levels and having either low or high LDL levels. The radioactivities in VLDL, intermediate density lipoprotein (IDL), LDL apoB, and urine were measured at intervals between 5 min and 6 days. Kinetic parameters for apoB were calculated in each baboon fed a chow diet or a high cholesterol, high fat diet (HCHF). VLDL apoB residence times were similar in the two groups of animals fed chow; they were increased by HCHF feeding in high LDL animals, but not in low LDL animals. Production rates of VLDL apoB were decreased by the HCHF diet in both high and low LDL animals. Most of the radioactivity from VLDL apoB was transferred to IDL. However, a greater proportion of radioactivity was removed directly from IDL apoB in low LDL animals than in high LDL animals, and only about one-third appeared in LDL. In high LDL animals, a greater proportion of this radioactivity was converted to LDL (61.4 +/- 7.2% in chow-fed animals and 49.2 +/- 10.9% in animals fed the HCHF diet; mean +/- SEM, n = 5). Production rates for LDL apoB were higher in high LDL animals than those in low LDL animals on both diets. The HCHF diet increased residence times of LDL apoB without changing production rates in both groups. VLDL apoB production was not sufficient to account for LDL apoB production in high LDL animals, a finding that suggested that a large amount of LDL apoB was derived from a source other than VLDL apoB in these animals.     

Effect of fasting on the composition of plasma lipoproteins in cholesterol-fed diabetic rabbits

Cholesterol-fat feeding is associated with unusual alterations in the composition of plasma lipoproteins in alloxan-diabetic rabbits. In the present study plasma lipoprotein lipid and apoprotein composition was studied before and after 48 hr of fasting in cholesterol-fed diabetic and control rabbits in order to further characterize these alterations. Compared with control rabbits, the diabetic rabbits had similar plasma cholesterol levels, but 100-fold higher triglyceride levels prior to fasting. These plasma lipids were distributed mainly to large, Sf greater than 400 plasma lipoproteins in the diabetic rabbits, and to beta-VLDL in control rabbits. Sf greater than 400 lipoproteins, VLDL, IDL, LDL, and HDL from diabetic rabbits had triglyceride as the predominant lipoprotein core lipid. Sf greater than 400 lipoproteins and VLDL from diabetic rabbits had lesser amount of apoprotein E, and greater amounts of apoproteins A-I, A-IV, and B-48 as percent of total apoprotein mass in comparison with control rabbits. Fasting reduced plasma triglyceride levels by 55% in diabetic rabbits. Sf greater than 400 lipoprotein and VLDL triglyceride content decreased but remained a major core lipid. Fasting eliminated apoproteins A-I and A-IV from Sf greater than 400 lipoproteins and VLDL, but had no significant effect on apoB-48 content. Insulin treatment of the diabetic rabbits reduced plasma triglyceride by approximately 90% resulting in cholesteryl ester-rich particles reassembling beta-VLDL both in the Sf greater than 400 lipoprotein and VLDL fractions. These results indicate that the alterations in plasma lipoproteins in cholesterol-fed diabetic rabbits result from the presence in the d less than 1.006 g/ml plasma lipoprotein class of partially metabolized, intestinally derived particles.     

Apolipoprotein B metabolism and the distribution of VLDL and LDL subfractions

Apolipoprotein B (apoB) metabolism was investigated in 20 men with plasma triglyceride 0.66-2.40 mmol/l and plasma cholesterol 3.95-6. 95 mmol/l. Kinetics of VLDL(1) (S(f) 60-400), VLDL(2) (S(f) 20-60), IDL (S(f) 12-20), and LDL (S(f) 0;-12) apoB were analyzed using a trideuterated leucine tracer and a multicompartmental model which allowed input into each fraction. VLDL(1) apoB production varied widely (from 5.4 to 26.6 mg/kg/d) as did VLDL(2) apoB production (from 0.18 to 8.4 mg/kg/d) but the two were not correlated. IDL plus LDL apoB direct production accounted for up to half of total apoB production and was inversely related to plasma triglyceride (r = -0.54, P = 0.009). Percent of direct apoB production into the IDL/LDL density range (r = 0.50, P < 0.02) was positively related to the LDL apoB fractional catabolic rate (FCR). Plasma triglyceride in these subjects was determined principally by VLDL(1) and VLDL(2) apoB fractional transfer rates (FTR), i.e., lipolysis. IDL apoB concentration was regulated mainly by the IDL to LDL FTR (r = -0.71, P < 0.0001). LDL apoB concentration correlated with VLDL(2) apoB production (r = 0.48, P = 0.018) and the LDL FCR (r = -0.77, P < 0. 001) but not with VLDL(1), IDL, or LDL apoB production. Subjects with predominantly small, dense LDL (pattern B) had lower VLDL(1) and VLDL(2) apoB FTRs, higher VLDL(2) apoB production, and a lower LDL apoB FCR than those with large LDL (pattern A). Thus, the metabolic conditions that favored appearance of small, dense LDL were diminished lipolysis of VLDL, resulting in a raised plasma triglyceride above the putative threshold of 1.5 mmol/l, and a prolonged residence time for LDL. This latter condition presumably permitted sufficient time for the processes of lipid exchange and lipolysis to generate small LDL particles.     

Effects of the acute phase response on the concentration and density distribution of plasma lipids and apolipoproteins

Longitudinal studies were carried out in the rabbit model to determine alterations in the concentration and density distribution of plasma lipids and apolipoproteins during the acute phase response (APR) characterized by elevated levels of C-reactive protein (CRP) and serum amyloid A (SAA). Twelve hr after the intramuscular injection of croton oil, SAA was detectable in high density lipoprotein (HDL). At the height of the response (72 hr), HDL decreased while SAA became the major HDL apoprotein, up to 80% of the proteins in the higher density fractions. The SAA-enriched particles became denser (density of HDL3) but larger (size of HDL2), had slower electrophoretic mobility, and were depleted in apoA-I, cholesterol, triglyceride, and phospholipid. HDL-cholesterol decreased and was redistributed to other fractions while apoA-I disappeared from the circulation. During this time plasma triglycerides increased 6- to 10-fold while plasma cholesterol and phospholipids showed minimal changes. ApoB increased 5- to 6-fold while the apoB-containing particles shifted to higher density resulting in elevated IDL and then LDL during recovery. VLDL (d less than 1.006 g/ml) increased and acquired 30-40% of the plasma triglycerides, cholesterol, phospholipid, and apoB. SAA also increased in VLDL while apoE decreased.     

Impaired plasma cholesteryl ester transfer with accumulation of larger high density lipoproteins in some families of baboons (Papio sp.)

Baboons from some families have a higher concentration of plasma high density lipoproteins (HDL) on a chow diet and accumulate large HDL (HDL1) when challenged with a high cholesterol and high saturated fat (HCHF) diet. HDL1 from high HDL1 animals contained more (1.5-fold) cholesteryl ester than HDL (HDL2 + HDL3) from high or low HDL1 animals. HDL from high HDL1 baboons had lower triglyceride content than that from low HDL1 baboons. HDL3 or HDL labeled with [3H]cholesteryl linoleate was incubated with entire lipoprotein fraction (d less than 1.21 g/ml) or very low density lipoprotein + low density lipoprotein (VLDL + LDL) (d less than 1.045 g/ml) and with lipoprotein-deficient serum (LPDS), and the radioactive cholesteryl ester and mass floating at d 1.045 g/ml (VLDL + LDL) after the incubation was measured. The transfer of cholesteryl esters from either HDL or HDL3, prepared from plasma of high HDL1 animals fed chow or the HCHF diet, was slower than the transfer from either HDL or HDL3 of low HDL1 animals, regardless of the source of transfer activity or the ratio of LDL:HDL-protein used in the assay. Addition of HDL from high HDL1 baboons into an assay mixture of plasma components from low HDL1 baboons decreased the transfer of cholesteryl ester radioactivity and mass from HDL to VLDL and LDL. In addition to HDL, a fraction of intermediate density lipoprotein (IDL) and denser HDL were also effective in inhibiting the transfer. These observations suggest that accumulation of HDL1 in high HDL1 baboons fed an HCHF diet is associated with a slower transfer of cholesteryl esters from HDL to LDL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Omega-3 fatty acids alter lipoprotein subfraction distributions and the in vitro conversion of very low density lipoproteins to low density lipoproteins

The purpose of this study was to determine the effects of a fish oil concentrate (FOC) on the in vitro conversion of very low density lipoproteins (VLDL) to intermediate (IDL) and low density lipoproteins (LDL). Six hypertriglyceridemic patients were randomly allocated to receive either placebo (olive oil) or FOC (1 g/14 kg body weight/day) for 4 weeks in a crossover study with a 4-week washout period. The FOC provided 3 g of eicosapentaenoic + docosahexaenoic acid per 70 kg of body weight, and it lowered plasma triglyceride and VLDL cholesterol levels by 35% and 42%, respectively. Decreases in the largest particles (VLDL(1)) were primarily responsible, with no effect noted in smaller VLDL particles (VLDL(2) and VLDL(3)). The FOC increased LDL cholesterol levels by 25% (P < 0.06) but did not affect LDL particle size. VLDL(1) and VLDL(3) were incubated in vitro with human postheparin lipases. Although triglycerides from both types of VLDL were hydrolyzed to the same extent with both treatments, particles isolated during the FOC phase were more readily converted into IDL and LDL than were control particles. These data suggest that the marine omega3 fatty acids may enhance the propensity of VLDL to be converted to LDL, partly explaining the decreased VLDL and increased LDL levels in FOC-treated patients.     

Effects of dietary fats and cholesterol on liver lipid content and hepatic apolipoprotein A-I, B, and E and LDL receptor mRNA levels in cebus monkeys.

The effects of the long-term administration of the dietary fats coconut oil and corn oil at 31% of calories with or without 0.1% (wt/wt) dietary cholesterol on plasma lipoproteins, apolipoproteins (apo), hepatic lipid content, and hepatic apoA-I, apoB, apoE, and low density lipoprotein (LDL) receptor mRNA abundance were examined in 27 cebus monkeys. Relative to the corn oil-fed animals, no significant differences were noted in any of the parameters of the corn oil plus cholesterol-fed group. In animals fed coconut oil without cholesterol, significantly higher (P less than 0.05) plasma total cholesterol (145%), very low density lipoprotein (VLDL) + LDL (201%) and high density lipoprotein (HDL) (123%) cholesterol, apoA-I (103%), apoB (61%), and liver cholesteryl ester (263%) and triglyceride (325%) levels were noted, with no significant differences in mRNA levels relative to the corn oil only group. In animals fed coconut oil plus cholesterol, all plasma parameters were significantly higher (P less than 0.05), as were hepatic triglyceride (563%) and liver apoA-I (123%) and apoB (87%) mRNA levels relative to the corn oil only group, while hepatic LDL receptor mRNA (-29%) levels were significantly lower (P less than 0.05). Correlation coefficient analyses performed on pooled data demonstrated that liver triglyceride content was positively associated (P less than 0.05) with liver apoA-I and apoB mRNA levels and negatively associated (P less than 0.01) with hepatic LDL receptor mRNA levels. Liver free and esterified cholesterol levels were positively correlated (P less than 0.05) with liver apoE mRNA levels and negatively correlated (P less than 0.025) with liver LDL receptor mRNA levels. Interestingly, while a significant correlation (P less than 0.01) was noted between hepatic apoA-I mRNA abundance and plasma apoA-I levels, no such relationship was observed between liver apoB mRNA and plasma apoB levels, suggesting that the hepatic mRNA of apoA-I, but not that of apoB, is a major determinant of the circulating levels of the respective apolipoprotein. Our data indicate that a diet high in saturated fat and cholesterol may increase the accumulation of triglyceride and cholesterol in the liver, each resulting in the suppression of hepatic LDL receptor mRNA levels. We hypothesize that such elevations in hepatic lipid content differentially alter hepatic apoprotein mRNA levels, with triglyceride increasing hepatic mRNA concentrations for apoA-I and B and cholesterol elevating hepatic apoE mRNA abundance.     

Metabolsim of apoB and apoC lipoproteins in man: kinetic studies in normal and hyperlipoproteininemic subjects.

The kinetics of apolipoproteins B and C were studied in 14 normal and hyperlipoproteinemic subjects after injection of exogenously (125)I-labeled very low density lipoprotein (VLDL) particles. Plasma radioactivities of apoB and apoC were determined over a period of 4 days in VLDL (d < 1.006) and total radioactivity in intermediate (IDL) (1.006 < d < 1.019), low (LDL) (1.019 < d < 1.063), and high (HDL) (1.063 < d < 1.21) density lipoproteins. The data were analyzed by the use of a model, developed mostly from these data, with the following results. The VLDL particle undergoes a series of incremental density changes, most likely due to a number of delipidation steps, during which apoB stays with the particle until the density reaches the IDL range. There is, however, a loss of apoC associated with these delipidation steps. In our normal subjects, all IDL apoB eventually becomes LDL. In our hyperlipemic subjects some of the apoB on IDL is also degraded directly. The apoC lost by VLDL and IDL recycles to HDL, and most of it is picked up again by newly synthesized VLDL. There is a slowdown of the stepwise delipidation process in all hyperlipemic individuals studied. Three additional features became apparent in the type III subjects. First, there is a significant increase (a factor of 2 compared to normal) in the apoB synthesis rate by way of VLDL; second, there is an induced direct apoB synthesis pathway by way of IDL (and/or LDL); third, a bypass of the regular stepwise VLDL delipidation pathway is induced by which VLDL particles lose apoC but none of their apoB, thereby forming a new particle with metabolic properties similar to LDL, but with a density still in the VLDL density range. Two type III patients treated with nicotinic acid and clofibrate showed a sharp decrease in their VLDL apoB synthesis rates. This was somewhat compensated by an increased IDL apoB synthesis rate. A type I patient on a medium chain triglyceride diet also showed a number of metabolic changes, including reduced VLDL apoB synthesis and the induction of considerable IDL and/or LDL apoB synthesis.     

Effect of dietary fish oil, vitamin E, and probucol on renal injury in the rat

Dietary fish oil, vitamin E, and probucol have been considered in a variety of human and experimental models of kidney disease. Using subtotal nephrectomized cholesterol-fed rats as a model for progressive kidney disease, we examined the effect of 5% dietary fish oil, or a combination of 5% dietary fish oil with 500 IU vitamin E/kg diet or 1% probucol on renal injury. Three-month-old Sprague Dawley rats were fed a control diet (C group) or a cholesterol supplemented (2%) diet (Ch group) containing either fish oil (FO group) or fish oil plus vitamin E (FO+E group) or fish oil plus probucol (FO+P group). After 4 weeks of dietary treatment, the right kidney was electrocoagulated and the left kidney nephrectomized. After 8 weeks, 24-hour urine was collected before sacrifice. No effect of the dietary treatments was noted on serum creatinine, blood urea nitrogen, or proteinuria, except that proteinuria was highest in FO+P group. Rats receiving the cholesterol diets had higher serum low density lipoprotein (LDL) + very low density lipoprotein (VLDL) cholesterol (P < 0.05). In contrast, rats in the FO+P group had the lowest serum total cholesterol and LDL+VLDL cholesterol among all groups. The FO group had 26% lower kidney alpha-tocopherol concentrations than the C group. However, inclusion of vitamin E in the diet (FO+E group) increased the kidney alpha-tocopherol status to a level comparable to that in the C group, whereas inclusion of probucol in fish oil diet (FO+P group) did not improve the kidney alpha-tocopherol status. Rats fed the cholesterol diet had a 2.5-fold higher glomerular segmental sclerosis (GSS) score and 1.5-fold higher glomerular macrophage (GM) subpopulation than the C group. These effects of the cholesterol diet were ameliorated by a fish oil diet (FO group: GSS by 30%, GM by 24%). The inclusion of vitamin E in the fish oil diet (FO+E group) did not further improve the GSS score or GM subpopulation. However, inclusion of probucol in fish oil diet (FO+P group) lowered the GSS score by 73% and reduced GM subpopulation by 83% compared with the Ch group. These remarkable changes can be attributed to the powerful hypocholesterolemic activity of probucol. Our findings indicate that progression of glomerular sclerosis in the rat remnant kidney model of progressive kidney disease can be significantly modulated with fish oil treatment.     

Effects of high cholesterol diets on rat plasma lipoproteins and lipoprotein-cell interactions

High fat, high cholesterol diets do not produce atherosclerotic lesions in some animal species such as the rat; however, when combined with experimentally induced hypothyroidism, such diets do produce lesions. While the diets or hypothyroidism each induce significant alterations in plasma lipoproteins, the combination produces marked hypercholesterolemia. If the atherosclerosis is related to the hyperlipidemia, the combination regimen could be provoking changes in the structure or compositions of lipoproteins which are not noted with either regimen alone. To test this hypothesis, Sprague-Dawley male rats (approximately 250 g) were treated as follows: Diet(a) = chow + 5% lard and 0.3% Na taurocholate; Diet(b) = Diet(a) + 2% cholesterol; Diet(c) = Diet(b) + 0.1% propylthiouracil (PTU). The major findings were as follows. 1) With Diet(b), slow floating very low density lipoprotein (VLDL) (pre-beta) enriched in cholesteryl esters accumulated in plasma and low density lipoprotein (LDL) disappeared from its usual flotation position. 2) With Diet(c), changes in plasma concentration were more marked but were also qualitatively different. More VLDL accumulated, and distribution of VLDL was shifted toward even slower floating cholesteryl ester-rich particles. VLDL had "broad beta" mobility. Also, a beta-migrating intermediate density lipoprotein (IDL) population appeared. 3) Lipoprotein (d less than 1.019 g/ml) and zonal subfractions of d less than 1.019 g/ml lipoproteins (isolated from rats on cholesterol Diet (b] stimulated [3H]oleate incorporation into cholesteryl esters of fibroblasts and macrophages, while the d less than 1.019 g/ml fractions of 5% fat (Diet(a]-fed rats did not. 4) The major finding of this study was that identically prepared d less than 1.019 g/ml fractions of Chol + PTU-treated rats (Diet(c] were approximately 2.5-fold more stimulatory than the lipoproteins of cholesterol-fed rats. The results could not be explained by differences in cholesterol contents of the cholesterol-rich lipoproteins, but significant differences in the apoprotein compositions of the fraction were found which could be important. The most active fractions had higher apoBL/apoBS and apoE/apoC ratios than less active fractions. Thus, the combination regimen of cholesterol and PTU produced changes in lipoprotein structure and composition which enhanced the abilities of the lipoproteins to interact with cells. The results suggest that analysis of lipoprotein-cell interactions in vitro may be predictive of the atherogenic potential of lipoproteins in vivo and that euthyroidism in rat protects against atherogenic hyperlipidemia.     

Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol.

Male rats were fed a cholesterol-free diet or the same diet supplemented with either 0.05, 0.1, 0.25, 0.5, 1, or 2% C for 21 days to investigate the effects of cholesterol on secretion of very low density lipoprotein (VLDL). Cholesterol feeding increased plasma and hepatic concentrations of triglyceride (TG) and cholesteryl esters (CE) in a dose-dependent manner. Plasma VLDL and low density lipoprotein (LDL) lipids were elevated by cholesterol feeding, while the high density lipoprotein (HDL) lipids were reduced. The secretion of the VLDL by perfused livers from these cholesterol-fed rats was examined to establish the relationship between the accumulation of lipids in the liver and the concurrent hyperlipemia. Liver perfusions were carried out for 4 h with a medium containing bovine serum albumin (3% w/v), glucose (0.1% w/v), bovine erythrocytes (30% v/v), and a 10-mCi 3H2O initial pulse. Oleic acid was infused to maintain a concentration of 0.6 mM. Hepatic secretion of VLDL-TG, PL (phospholipid), free cholesterol (FC), and CE increased in proportion to dietary cholesterol and was maximal at 0.5% cholesterol in these experiments in which TG synthesis was stimulated by oleic acid. Secretion of VLDL protein and apoB by the perfused liver was also increased. The molar ratios of surface (sum of PL and cholesterol) to core (sum of TG and CE) lipid components of the secreted VLDL, regardless of cholesterol feeding, were the same, as were the mean diameters of the secreted particles. The molar ratios of surface to core lipid of VLDL isolated from the plasma also were not affected by cholesterol feeding. During perfusion with oleic acid of livers from the rats fed the higher levels of cholesterol, the hepatic concentration of CE decreased, while the level of TG was not changed. We conclude that the hypercholesterolemia and hypertriglyceridemia that occur in vivo from cholesterol feeding, concurrent with accumulation of CE and TG in the liver, must result, in part, from increased hepatic secretion of all VLDL lipids and apoB. The VLDL particles produced by the liver of the cholesterol-fed rat are assembled without modification of the surface lipid ratios (PL/FC), but contain a greater proportion of cholesteryl esters compared to triglyceride in the core, because of the stimulated transport of CE from the expanded pool in the liver.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lovastatin therapy reduces low density lipoprotein apoB levels in subjects with combined hyperlipidemia by reducing the production of apoB-containing lipoproteins: implications for the pathophysiology of apoB production

We investigated the metabolism of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and low density lipoprotein (LDL) apolipoprotein B (apoB) in seven patients with combined hyperlipidemia (CHL), using 125I-labeled VLDL and 131I-labeled LDL and compartmental modeling, before and during lovastatin treatment. Lovastatin therapy significantly reduced plasma levels of LDL cholesterol (142 vs 93 mg/dl, P less than 0.0005) and apoB (1328 vs 797 micrograms/ml, P less than 0.001). Before treatment, CHL patients had high production rates (PR) of LDL apoB. Three-fourths of this LDL apoB flux was derived from sources other than circulating VLDL and was, therefore, defined as "cold" LDL apoB flux. Compared to baseline, treatment with lovastatin was associated with a significant reduction in the total rate of entry of apoB-containing lipoproteins into plasma in all seven CHL subjects (40.7 vs. 25.7 mg/kg.day, P less than 0.003). This reduction was associated with a fall in total LDL apoB PR and in "cold" LDL apoB PR in six out of seven CHL subjects. VLDL apoB PR fell in five out of seven CHL subjects. Treatment with lovastatin did not significantly alter VLDL apoB conversion to LDL apoB or LDL apoB fractional catabolic rate (FCR) in CHL patients. In three patients with familial hypercholesterolemia who were studied for comparison, lovastatin treatment increased LDL apoB FCR but did not consistently alter LDL apoB PR. We conclude that lovastatin lowers LDL cholesterol and apoB concentrations in CHL patients by reducing the rate of entry of apoB-containing lipoproteins into plasma, either as VLDL or as directly secreted LDL.     

Dietary oxidized cholesterol decreases expression of hepatic microsomal triglyceride transfer protein in rats

The aim of this study was to compare the effects of dietary oxidized cholesterol and pure cholesterol on plasma and very low density lipoprotein (VLDL) lipids and on some parameters of VLDL assembly and secretion in rats fed two different dietary fats. Four groups of male growing Sprague-Dawley rats were fed diets containing pure or oxidized cholesterol (5 g/kg diet) with either coconut oil or salmon oil as dietary fat (100 g/kg diet) for 35 days. Rats fed oxidized cholesterol supplemented diets had significantly lower concentrations of triglycerides and cholesterol in plasma and VLDL than rats fed pure cholesterol supplemented diets irrespective of the type of fat. In addition, rats fed oxidized cholesterol supplemented diets had significantly lower relative concentrations of microsomal triglyceride transfer protein messenger ribonucleic acid (mRNA) than rats fed pure cholesterol supplemented diets. In contrast, hepatic lipid concentrations and the relative concentration of apolipoprotein B mRNA were not influenced by the dietary factors investigated. Parameters of hepatic lipogenesis (relative mRNA concentration of sterol regulatory element binding protein-1c and activity of glucose-6-phosphat dehydrogenase) were significantly reduced by feeding fish oil compared to coconut oil, but were not affected by the type of cholesterol. In conclusion, the data of this study suggest, that dietary oxidized cholesterol affects VLDL assembly and/or secretion by reducing the synthesis of MTP but not by impairing hepatic lipogenesis or synthesis of apolipoprotein B.     

Resistant starch fraction prepared from kintoki bean affects gene expression of genes associated with cholesterol metabolism in rats

Feeding rats beans with resistant starch reduces the serum cholesterol concentration; however, the mechanism is not fully understood. We examined the effects of resistant starch of kintoki (Phaseolus vulgaris, variety) bean on serum cholesterol and hepatic mRNAs in rats. Male F344/Du Crj rats were fed a cholesterol-free diet either with 5 g of cellulose powder (control)/100 g or 5 g of pancreatin-resistant fraction prepared from kintoki bean (kintoki)/100 g diet for 4 weeks. There were no differences in the body weight gain, food intake, liver weight, and mass of cecum contents between the groups. Serum total cholesterol, very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) + low density lipoprotein (LDL)-cholesterol, and high density lipoprotein (HDL)-cholesterol levels in the kintoki group were significantly (at least P < 0.05) lower than in the control group throughout the feeding period. There was no difference in the serum triglyceride concentration between two groups throughout the feeding period. Total hepatic cholesterol in the control group was significantly (P < 0.01) lower than in the kintoki groups. Fecal bile acid, cecal acetate, propionate and n-butyrate concentrations in the kintoki group all were significantly (P < 0.05) higher than in the control group. Likewise, hepatic cholesterol 7alpha-hydroxylase, LDL receptor, and SR-B1 mRNA levels in the kintoki group were significantly (P < 0.05) higher than in the control group. The results suggest that resistant starch of kintoki bean reduces serum cholesterol level by increasing hepatic LDL receptor, SR-B1, and cholesterol 7alpha-hydroxylase mRNAs.     

Isopropanol precipitation method for the determination of apolipoprotein B specific activity and plasma concentrations during metabolic studies of very low density lipoprotein and low density lipoprotein apolipoprotein B

A method has been described for the measurement of apoB concentration and specific activity in very low density lipoprotein (VLDL) and low density lipoprotein (LDL) during metabolic studies. For measurement of specific activity, apoB was separated from other apolipoproteins and lipids by precipitation in, and subsequent washing with, isopropanol. For determination of apoB concentration, equal volumes of lipoprotein and isopropanol were mixed, and the protein content of the apoB precipitate was measured by the difference between total lipoprotein protein and the protein measured in the supernatant. Evidence that there was no apoB solubilization in isopropanol and that precipitated apoB was virtually free of soluble apolipoproteins was obtained by electrophoresis. Lipid contamination of the apoB precipitate was less than 1%. The methods were applicable to VLDL, intermediate density lipoprotein (IDL), and LDL from normolipemic patients with protein concentrations between 187 micrograms/ml and 1898 micrograms/ml. The data obtained using isopropanol were highly correlated with those using tetramethylurea, and recoveries of apoB were similar. Furthermore, the isopropanol method has been demonstrated to yield consistent data for apoB specific activities in a study of VLDL, IDL, and LDL metabolism.     

Evaluation of the use of serum lathosterol concentration to assess whole-body cholesterol synthesis in rabbits.

Serum lathosterol concentration in rabbits was assessed as a possible indicator of whole-body cholesterol synthesis. In random-bred New Zealand White (NZW) rabbits fed a control diet or a diet containing either cholesterol, simvastatin, or cholestyramine, neither serum lathosterol concentration nor the serum lathosterol:total cholesterol ratio systematically corresponded with the anticipated rate of cholesterol synthesis. In control rabbits and those fed simvastatin or cholestyramine, whole-body cholesterol synthesis, which was calculated from the sterol balance, was correlated with serum lathosterol concentration when expressed relative to cholesterol in very low, intermediate, and low density lipoproteins (VLDL + IDL + LDL) (r = 0.61; n = 23; P = 0.002). The low correlation coefficient indicates that the predictive value of the lathosterol: (VLDL + IDL + LDL) cholesterol ratio is limited when applied to individual rabbits. Cholesterol and simvastatin feeding reduced the group mean serum lathosterol:(VLDL + IDL + LDL) cholesterol ratio, whereas cholestyramine in the diet raised the group mean ratio in the NZW rabbits. We conclude that the serum lathosterol:(VLDL + IDL + LDL) cholesterol ratio may be an indicator of group mean rates of whole-body cholesterol synthesis in rabbits but may not yield reliable information on individual rabbits. The lathosterol:(VLDL + IDL + LDL) cholesterol ratio predicted that in hyperresponsive inbred rabbits, showing an excessive hypercholesterolemia after cholesterol feeding, baseline whole-body cholesterol synthesis is lower than in hyporesponsive rabbits. Addition of cholesterol to the diet caused a reduction of predicted cholesterol synthesis in hypo- but not in hyper-responsive rabbits.