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Guinea pigs were fed 15% (w/W) fat, high in lauric and myristic acids, a diet known to produce hypercholesterolemia in these animals. The diet was given alone or in combination with four doses of atorvastatin equivalent to 1, 3, 10, and 20 mg/kg per day. Atorvastatin reduced plasma LDL cholesterol concentrations by 46, 50, 53, and 70%, respectively (P < 0.001). Plasma apoB concentrations were reduced by atorvastatin (P < 0.001) and compositional changes occurred in VLDL and LDL with reductions of the relative proportion of cholesteryl ester and increases in triacylglycerol. A reduction in hepatic cholesteryl ester (66%) was observed only with the highest atorvastatin dose (20 mg/kg per day) while microsomal cholesterol was reduced by 30% with 3-20 mg/kg per day. Hepatic ACAT activity was down-regulated and apoB/E receptor number was increased by atorvastatin. In contrast, HMG-CoA reductase activity and cholesterol 7 alpha-hydroxylase were not affected by the drug. VLDL apoB secretion rates were decreased by atorvastatin treatment 59 and 76% with 3 and 20 mg/kg per day, respectively. Nascent VLDL particles were larger after drug treatment, showing an increased number in triacylglycerol molecules. These results support the hypothesis that the plasma LDL lowering induced by atorvastatin is due to a decreased secretion of apoB in combination with an increase of hepatic apoB/E receptors.

A human cDNA clone (K1) was recently isolated that encodes functional acyl-CoA:cholesterol acyltransferase (ACAT) protein (Chang et al. J. Biol. Chem. 1993. 268: 20747-20755). We used the K1 clone to screen a rabbit liver cDNA library and isolated a 919 base pair partial rabbit cDNA (ACAT14b) that was greater than 90% homologous with the human nucleotide sequence. Northern blotting using the rabbit ACAT cDNA14b revealed the existence of at least six related mRNA species (ranging from 6.2 to 1.7 kb) in various rabbit tissues. Using an RNAse protection assay, ACAT mRNA14b was detected in twelve separate rabbit organs. Adrenal gland contained the highest concentrations of ACAT mRNA14b (per microgram of total RNA) being 20-, 30-, and 50-fold higher than small intestine, aorta, and liver, respectively. Additional studies with isolated liver cell populations revealed that rabbit hepatic nonparenchymal cells contained 30-fold more ACAT mRNA14b (per microgram of total RNA) than parenchymal cells. To determine whether ACAT mRNA14b levels are regulated in vivo, rabbits were fed for 4 weeks a high fat/high cholesterol diet (HFHC; 0.5% cholesterol, 3% coconut oil, 3% peanut oil) at which point they were either kept for an additional 4 weeks on the HFHC-diet or switched to the HFHC-diet plus CI-976 (50 mg/kg), a potent and specific ACAT inhibitor; another group of rabbits was fed a chow diet for the entire 8 weeks. The HFHC-diet caused a 2- and 3-fold increase in hepatic and aortic ACAT mRNA14b levels, respectively, in comparison to chow-fed animals; there was no change in adrenal or small intestine levels. CI-976 treatment lowered ACAT mRNA14b levels by 60% and 40% in liver and aorta, respectively, in comparison to the HFHC controls; again there was no change in adrenal or small intestine levels. These data indicate that ACAT mRNA14b levels increase in a tissue specific manner in response to dietary fat and cholesterol.

Rat mesenteric lymph contains all serum apolipoproteins. However, it is uncertain whether some of these apolipoproteins are derived from intestinal synthesis or are transferred from plasma. We compared lymph apolipoprotein composition, concentrations, and transport rates in normal rats and in rats treated with pharmacologic doses of ethinyl estradiol which have negligible concentrations of serum lipids and apolipoproteins. Lymph apolipoproteins were examined before and after duodenal lipid infusion. Lymph d less than 1.006 and 1.006-1.21 g/ml lipoproteins were isolated and SDS-electrophoresis was performed using 10 and 3.5% polyacrylamide. During lipid absorption, lymph flow increased in control but not in treated rats. Control lymph contained all major apolipoproteins, but lymph from ethinyl estradiol-treated rats contained only apoB, A-I, and A-IV. Two apoB bands were noted on 3.5% gels in control lymph, but only the lower molecular weight protein was found in lymph from ethinyl estradiol-treated rats. In control rats, transport rates for apoA-I, A-IV, E, and C proteins increased during lipid absorption, but only in the case of A-IV was this a reflection of increased apolipoprotein concentration and not the enhanced lymph flow. In ethinyl estradiol-treated rats only the A-IV transport rate increased due to lipid infusion. It is concluded that in the ethinyl estradiol-treated rat 1) the intestine does not synthesize apoE, C, or the high molecular weight apoB; 2) lymphatic output of A-IV is predominantly increased during lipid absorption; and 3) since plasma apolipoprotein concentrations are negligible, lymph lipoproteins from ethinyl estradiol-treated rats may represent a close approximation to nascent particles of intestinal origin.

A reliable method for the separation of free and esterified cholesterol in adipocyte extracts is described. The procedure uses Sephadex LH-20, a lipophilic dextran gel, with a solvent system of chloroform-hexane 55:45 (v/v). Interference by excess triglyceride, such as that encountered in adipocyte total lipid extracts, was not observed, and overall recovery of both sterols exceeded 98%.

Rats were fed either rat chow (control), chow + 20% olive oil (olive oil), or chow + 20% olive oil + 2% cholesterol (olive oil/cholesterol) as a single meal to study the short-term effects of fat and the above combination of fat/cholesterol-containing diets on plasma apoB concentration and its influence on the distribution of apoB subspecies. Rats were given their meals and allowed to consume them ad libitum until they were killed, 3 hr or 9 hr afterwards. Three hours after feeding, serum triglyceride concentrations were increased to the same extent in both the olive oil and olive oil/cholesterol-fed rats as compared with concentrations in control rats, but serum apoB concentrations did not differ among the groups. Nine hours after feeding, serum triglyceride concentrations were still equally elevated in both experimental groups; however, in the olive oil/cholesterol-fed rats, total serum apoB as well as total serum cholesterol were increased above both the control and olive oil groups. In addition, the d less than 1.21 g/ml lipoprotein apoBl/apoBh ratio of the olive oil/cholesterol-fed rats was greatly increased at 9 hr, whereas apoBl/apoBh ratio in the d less than 1.21 g/ml fraction of the olive oil group was unchanged, despite the increase in plasma triglyceride concentration. In the olive oil/cholesterol-fed rats at 9 hr, cholesterol, total apoB, apoBl, and apoBh of both VLDL and IDL were greater than in the control or olive oil rats. In d less than 1.21 g/ml lipoproteins, VLDL, and IDL, the increases in apoBl concentrations were of a greater magnitude than the increases in apoBh.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Adipose tissue in man is a major site for cholesterol storage. In obesity over half of total body cholesterol may reside within this tissue; however, relatively little attention has been directed toward understanding the cholesterol metabolism and its relationship to whole body cholesterol homeostasis in this tissue. In this review the factors which influence cholesterol storage are discussed, with particular emphasis on the effects of diet and drug treatment in both animals and man. The uptake, synthesis, and mobilization of adipose tissue cholesterol appears to be mediated and/or regulated, as in other tissues, by the plasma lipoproteins, and these processes are examined with regard to both normal and pathologic states.

The distribution, chemical, and apoprotein composition of plasma and peripheral lymph lipoproteins were compared in control and cholesterol-fed dogs. In both groups of animals, the agarose electrophoretic patterns of plasma and lymph lipoproteins were similar. In hypercholesterolemic dogs, beta-very low density lipoprotein, beta-migrating intermediate density lipoprotein, and HDLc were major components both in plasma and lymph, providing evidence for a potential interaction of these atherogenic particles with macrophages and other peripheral cells. The chemical composition and physical appearance of peripheral lymph HDL was markedly different from that of plasma HDL (high density lipoprotein), especially in the cholesterol-fed animals. Lymph HDL had a higher cholesterol to protein ratio and a markedly increased free cholesterol content (free cholesterol to cholesteryl ester ratio of 1.7 as opposed to 0.2 in plasma HDL in cholesterol-fed animals). The phospholipid content of lymph HDL was higher than that of plasma HDL, while the protein content was lower. A significant proportion of lymph HDL obtained from cholesterol-fed dogs was in the form of disc-shaped particles stacked in rouleau structures. Changes in plasma apolipoprotein concentrations due to cholesterol feeding were reflected in peripheral lymph to different degrees, depending largely on the relative size of the lipoproteins containing the individual lipoproteins. A considerable enrichment of lymph HDL with apoE and apoA-IV was observed by both immunochemical and electrophoretic methods. In lymph HDL from control and cholesterol-fed dogs, the apoE/apoA-I and apoA-IV/apoA-I ratios were several-fold elevated, compared to those of plasma HDL. It is concluded, therefore, that during cholesterol feeding a substantial portion of interstitial HDL is assembled de novo in the periphery as a crucial stage of reverse cholesterol transport to the liver. It is likely that further modification occurs upon entry to plasma and exposure to lecithin:cholesterol acyltransferase, possibly leading to generation of HDLc. Alternatively, these particles may be directly and rapidly removed by the liver.