Hepatic binding and internalization of low density lipoprotein-gold conjugates in rats treated with 17 alpha-ethinylestradiol.

Receptor-mediated hepatic uptake of low density lipoproteins (LDL) conjugated to colloidal gold was studied by perfusion of livers from rats treated for 5 d with 17 alpha-ethinylestradiol. Estrogen treatment resulted in a marked decrease in serum lipid and lipoprotein concentrations. After 15 min of perfusion the conjugate was bound to the hepatic microvilli of both control and estrogen-treated rats; the estrogen-treated rats showed an 8- to 11-fold greater number of membrane-bound conjugates. The conjugates were bound to the membrane receptor by the LDL particle because the gold granules were regularly displaced from the membrane by 20 +/- 3.2 nm, the diameter of LDL. Internalization of the conjugate, evident by gold particles in multivesicular bodies, occurred at coated pits at the base of the microvillus where coated vesicles containing a single gold-LDL conjugate were released. After 1 h of perfusion, the livers from the estrogen-treated rats showed all phases of endocytosis and incorporation into multivesicular bodies of the conjugate. After 2 h of perfusion, there was congregation of gold-labeled lysosomes near the bile canaliculi. Gold-LDL conjugates were also observed to bind and be internalized by Kupffer cells and sinusoidal endothelium. These findings indicate that estrogen treatment induces hepatic receptors for LDL. The catabolic pathway of binding and endocytosis of the conjugate is similar to that seen in fibroblasts, although slower. Because gold-LDL conjugates were also present in the Kupffer and endothelial cells, the uptake of LDL by the liver involves the participation of more than a single cell type.

An MTP inhibitor that normalizes atherogenic lipoprotein levels in WHHL rabbits.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

The effect of pravastatin on serum cholesterol levels in hypercholesterolemic diabetic rabbits.

Diabetes mellitus is associated with hyperlipidemia and increased risk of atherosclerosis. A diabetic animal model has been developed to study the effect of treatment with pravastatin, a potent HMG CoA reductase inhibitor, on plasma lipoprotein levels. Hypercholesterolemia was induced in alloxan diabetic and control rabbits by feeding a diet containing 25% casein and 10% hydrogenated coconut oil for 8 weeks. Feeding the casein-coconut oil diet to the diabetic group resulted in a 5-fold increase in serum cholesterol levels, which was not statistically different from the nondiabetic group fed this diet. However, in the diabetic group, there was more cholesterol in the VLDL fraction and less in LDL as compared to the nondiabetic group. Serum triacylglycerol levels in the diabetic rabbits were variable and ranged from 58-943 mg/dl. The diabetic and nondiabetic animals were then treated with pravastatin at a dose of 10 mg/kg per day for 21 days. In the nondiabetic group, pravastatin treatment significantly lowered serum and LDL cholesterol concentrations by 28.5% (52.3 mg/dl, P less than 0.05) and 36.2% (40.7 mg/dl, P less than 0.05) respectively, relative to the placebo group. Serum and VLDL triacylglycerol levels in the nondiabetic group were also significantly decreased following pravastatin treatment. In the diabetic group, serum and LDL cholesterol levels were decreased by 37.0% (69.1 mg/dl, P less than 0.05) and 52.7% (32.1 mg/dl, P less than 0.01), respectively, relative to the diabetics given the placebo. Pravastatin treatment did not adversely affect serum glucose levels. Thus, pravastatin treatment was effective in controlling the hypercholesterolemia present in these diabetic animals.

Effects of hyperlipidemias in hamsters on lipid transfer protein activity and unidirectional cholesteryl ester transfer in plasma.

Experiments were performed to characterize plasma lipid transfer protein activity (LTA), and the rate of [3H]CE transfer from HDL to lower density lipoproteins in plasma of hamsters. Compared to rabbits, hamster plasma has about one-tenth the level of d greater than 1.21 LTA but a relatively high level of VLDL-triacylglycerols, and a higher fractional rate of HDL-[3H]CE transfer in plasma (in vitro) than predicted by the d greater than 1.21 LTA. Like the rat, hamster plasma contains an inhibitor(s) of LTA; the level of the inhibitor activity in d greater than 1.21 g/ml plasma was similar in normal and hyperlipoproteinemic hamsters. Hypertriglyceridemia in sucrose-fed hamsters did not affect LTA, cholesteryl ester transfer or the plasma level of HDL-CE. However, a comparable degree of hypercholesterolemia was associated with a 122% increase in plasma d greater than 1.21 LTA and a 63% increase in the fractional rate of [3H]CE transfer from HDL to lower density lipoproteins in plasma. Cholesterol feeding in hamsters was associated with increased plasma levels of LDL-cholesterol and, to a lesser extent, with VLDL- and IDL-cholesterol.

The uptake of the apoprotein and cholesteryl ester of high-density lipoproteins by the perfused rat liver.

The uptake of the 125I-labeled apolipoprotein and 3H-labeled cholesteryl ester components of rat apolipoprotein E-deficient HDL by the perfused liver was studied. The uptake of the cholesteryl ester moiety was 4-fold higher than that of apolipoprotein. The concentration-dependent uptake of labeled protein was saturable and competed for by an excess of unlabeled HDL. The uptake of cholesteryl ester was not saturable over the concentration range studied. In the presence of a 50-fold excess of unlabeled HDL, the uptake of both radiolabeled components was decreased by over 75%, indicating that three-quarters of the hepatic uptake of HDL is by a receptor-mediated process. After 15 min of perfusion, 37% of the apolipoprotein radioactivity that was initially bound at 5 min was released into the perfusate as a more dense particle. After 5, 15, 30 and 60 min of perfusion the subcellular distribution of the apolipoprotein and cholesteryl ester components was analyzed by Percoll density gradient centrifugation. Over the 60 min period, there appeared to be transfer of radioactivity from the plasma membrane fraction to the lysosomal fraction. However, the internalization and degradation of cholesteryl ester was more rapid than that of the apolipoprotein. Our findings indicate that there is preferential uptake of HDL cholesteryl ester relative to protein by the liver and that the internalization of these components may occur independently.

The activity and sedimentation properties of the aminoacyl-tRNA synthetases of rat skeletal muscle.

The aminoacyl-tRNA synthetases of the postribosomal supernatant fraction of rat skeletal muscle were characterized by their activity and sedimentation properties. The synthetases of muscle were compared with those of liver in terms of these parameters. Extraction of the synthetases of muscle with a buffer containing 4 mM adenosine triphosphate (ATP) resulted in a 50--100% increase in the activities of glutaminyl-, glutamyl-, isoleucyl-, leucyl-, lysyl-, methionyl-, and prolyl-tRNA synthetases in the postribosomal fraction, over those activities extracted in the absence of ATP. This effect of ATP was specific for those synthetases which sedimented as particulate elements in sucrose gradients, and appeared to be unique to muscle. The individual synthetase activities of muscle, except alanyl-, leucyl-, and valyl-tRNA synthetases, were aprrox. 25% of the corresponding synthetase activities of liver. Sucrose density gradient analysis of the postribosomal fraction of muscle and liver revealed that the sedimentation profiles of the synthetases of the two tissues were similar, with nine synthetase activities sedimenting as large particulate entities at 18 S. The findings suggest that the particulate forms of the synthetases reflect true association of the enzymes with a high molecular weight cellular component common to both tissues.

Serum lipoproteins of diabetic rats fed a high cholesterol diet.

Feeding a diet containing 2% cholesterol and 1% cholic acid (wt/wt) to rats made diabetic by administration of streptozotocin (45 mg/kg) produced marked hypercholesterolemia characterized by high concentrations of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL) and a reduction in concentration of high density lipoproteins (HDL). The VLDL was unique in that it contained apo A-I and apo A-IV in addition to its usual complement of apoproteins: apo B, apo E, and the C apoproteins. IDL had a similar apoprotein composition. The HDL from these rats was deficient in apo E. Nondiabetic rats fed the same diet exhibited similar qualitative changes in lipoprotein concentration and composition but with lesser increases in VLDL and IDL concentrations. The altered apoprotein composition suggested that the hyperlipoproteinemia associated with cholesterol feeding in the rat is due to an inadequate rate of removal of lipoproteins of intestinal origin, and that this is greatly exacerbated by diabetes.

Expression of human hepatic glucokinase in transgenic mice liver results in decreased glucose levels and reduced body weight.

Glucokinase is the predominant hexokinase in pancreatic beta-cells and liver parenchymal cells and functions as a critical component of the glucose-sensing apparatus in these glucose-responsive cell types. In the beta-cells, the sensing leads to insulin secretion, while the role in hepatocytes is thought to be in hepatic glucose uptake. To determine the physiological response to an increase in hepatic glucokinase expression, transgenic mice expressing the human hepatic glucokinase gene under the control of a liver-specific human apolipoprotein A-I gene enhancer were generated. Transgenic mice had twofold higher total fasting hepatic glucokinase mRNA, which resulted in a modest 20% increase in fasting glucokinase activity. These animals showed lower fasting plasma glucose, insulin, and lactate levels and improved tolerance to glucose. In addition, glucokinase transgenic animals weighed less and had lower BMI than nontransgenic animals. Thus, glucokinase transgenic animals demonstrate that a modest change in hepatic glucokinase activity enhances the metabolism of glucose.

Sinusoidal endothelial endocytosis of low density lipoprotein-gold conjugates in perfused livers of ethinyl-estradiol treated rats.

We examined endocytosis of low-density lipoproteins (LDL) conjugated to colloidal gold by the sinusoidal endothelium in perfused livers of 17 alpha-ethinyl estradiol-treated rats. After 15 min of perfusion, the gold-LDL was randomly bound at the endothelial surface, but internalized only at coated pits. Uptake of the conjugate was to electron-lucid vacuoles. After 1 h of perfusion, there was a progressive accumulation of gold in organelles resembling lysosomes, with further accumulation seen at 2 h perfusion. Uptake of the conjugate was equivalent to the rate of 125I-LDL and competitively inhibited by a 20-fold excess of free LDL. These results suggest that a specific endocytotic pathway for LDL is present in the sinusoidal endothelium in the estrogen-hypolipidemic state.

Beta 3-adrenergic receptor-mediated lipolysis and oxygen consumption in brown adipocytes from cynomolgus monkeys.

Primary adipocytes were isolated from axillary brown adipose tissue from adult cynomolgus monkeys. That this tissue contained brown adipocytes was verified by morphological examination and by demonstrating the presence of uncoupling protein messenger ribonucleic acid in the isolated adipocytes. The contributions of beta 1-, beta 2-, and beta 3-adrenergic receptors (AR) to lipolysis and oxygen consumption of isolated brown adipocytes were determined after agonist stimulation. Dose responses were determined using isoproterenol (a nonselective beta-AR agonist), denopamine (beta 1-AR agonist), procaterol (beta 2-AR agonist), and CGP12177A (beta 1- and beta 2-AR antagonist, beta 3-AR agonist). Isoproterenol, denopamine, and procaterol stimulated lipolysis with EC50 values of 4,500, and 83 nmol/L, respectively. Intrinsic activities (relative to isoproterenol maxima) were 100%, 74%, and 59%, respectively. The presence of beta 3-ARs coupled to lipolysis was demonstrated by the activity of CGP12177A (EC50 = 1.6 mumol/L; intrinsic activity = 62%). Isoproterenol stimulated oxygen consumption of brown adipocytes by 75-100% above the basal rate, with an EC50 of 1 mumol/L. Denopamine, procaterol, and CGP12177A stimulated oxygen consumption at a concentration of 100 mumol/L. These results demonstrate that all three beta-adrenergic receptor subtypes are coupled to lipolysis and oxygen consumption in brown adipocytes from cynomolgus monkeys.

Hyperlipoproteinemia in spontaneously diabetic guinea pigs.

A colony of Hartley guinea pigs that exhibit hyperglycemia, glucosuria, and hypertriglyceridemia characteristic of human diabetes mellitus was developed. Initially, a group of guinea pigs that had normal serum glucose concentrations (less than or equal to 200 mg/dL of serum) at 3 to 4 weeks of age was obtained; however, in some of the animals progressively severe hyperglycemia (300 to 500 mg/dL of serum) and glucosuria (greater than 2 g of glucose/24 h) occurred as the animals matured. In addition, the animals exhibiting hyperglycemia and glucosuria had plasma insulin concentrations that were similar to those animals that were not hyperglycemic. The diabetic animals were found to be hypertriglyceridemic, with plasma triglyceride levels of 140 to 290 mg/dL at four months of age. Nondiabetic animals (plasma glucose concentration of less than or equal to 200 mg/dL and no glucosuria) had plasma triglyceride concentrations between 37 and 76 mg/dL. Lipoprotein analysis of plasma from nondiabetic and diabetic animals indicated that the diabetics had a fourfold increase in VLDL triglyceride and protein concentrations. The VLDL had an abnormal apolipoprotein composition and had reduced levels of apoprotein-E. The progeny from the mating of diabetic males and females also exhibited the diabetic trait, suggesting that the origin of the disease is genetic. This colony of guinea pigs is being further investigated as a suitable model for the study of the hyperlipoproteinemia of human noninsulin-dependent diabetes mellitus.

Determinants of the uptake of very low density lipoprotein remnants by the perfused rat liver.

The receptor-mediated uptake of very low density lipoprotein (VLDL) remnants by the rat liver was studied. Livers were perfused with native 125I-VLDL remnants, radiolabeled apo E-deficient remnants, and radiolabeled remnants that contained reductively methylated apo B and unmodified apo E. The specific uptake of the apo E-deficient remnants was 20% of that for the native remnants, whereas the specific uptake of the remnants containing unreactive apo B was 78% of the control value. This suggests that the apo E of VLDL remnants is the principal ligand for binding to the receptor, and in the absence of apo E, apo B may participate in binding. This conclusion is supported by the finding that dimyristoyl phosphatidylcholine (DMPC)- apo E complexes were effective in competing for the hepatic uptake of 125I-VLDL remnants. The intracellular distribution of radioactivity was analyzed by Percoll density gradient centrifugation. At five minutes after perfusion, radioactivity was associated with the plasma membrane and lysosomal fractions, and at 30 minutes most of the radioactivity was associated with the lysosomal fraction. Binding and internalization of VLDL remnants was also directly visualized by electron microscopy. Internalization proceeded by coated pit-coated vesicle formation with subsequent delivery to lysosomes. Our findings demonstrate that the apo E of VLDL remnants mediates binding to the hepatic receptor and that the internalization and degradation of VLDL remnants is by a similar pathway to that previously described for LDL.

Bile acid sequestrants: their use in combination with other lipid-lowering agents.

Several potent drugs are currently available for the treatment of hyperlipidemia. These pharmacotherapies include bile acid sequestrants, HMG-CoA reductase inhibitors (commonly referred to as 'statins'), fibrates and nicotinic acid. Combination therapy to reduce plasma cholesterol levels in hyperlipidemic patients is becoming more popular, as concerns about toxicity resulting from the long-term used of systemically acting drugs are raised. One of the more widespread combinations used to reduce plasma cholesterol levels is that of a bile acid sequestrant and a statin. Combinations of bile acid sequestrants with either niacin or fibrates have been found to be very effective in patients with mixed hyperlipidemia, since the triglyceride-lowering effect of niacin or a fibrate complements the cholesterol-lowering effect of the sequestrant. One major advantage of combination therapy is the ability to reduce the dose of each component to more tolerable levels for the patient. An additional advantage is the reduced cost to achieve the cholesterol reduction goal, as the dose response is relatively good at greatly reduced doses for each component.

Ultrastructure of hepatic cholesterol crystals in the hypercholesterolemic - diabetic rat.

The cellular morphology of lipid accumulation in the liver was examined in normal rats fed a diet containing cholesterol and cholic acid, and streptozotocin-diabetic rats fed the same diet. The cholesterol-fed non diabetic rats displayed moderate hypercholesterolemia (average cholesterol 317 mg/dl) whereas the cholesterol-fed diabetic rats exhibited severe hypercholesterolemia (cholesterol greater than 1300 mg/dl). Ultrastructural studies were performed on hepatic tissues following in situ fixation and water soluble embedment, which were used to reduce lipid extraction and minimize structural distortions. Although both groups exhibited hepatocyte lipid droplets, the accumulation was markedly accentuated in the diabetic animals. The Kupffer cells of the diabetic animals contained cytosolic lipid crystals that were membrane delimited and showed lattice ordering 3.9 +/- 2.2 nm periodicity. These findings suggest that cholesteryl ester crystals of the cholesteric phase, similar to those found in atherosclerotic lesions, may form in other cellular foci exposed to abnormally high plasma lipid levels.

The uptake of chylomicron remnants and very low density lipoprotein remnants by the perfused rat liver.

The regulation of the hepatic uptake of chylomicron remnants and very-low-density lipoprotein (VLDL) remnants was studied in the rat using a nonrecirculating liver perfusion system. The hepatic removal of remnant lipoproteins was shown to be by receptor-mediated processes since the concentration-dependent uptake was saturable and reductive methylation of the particles reduced the uptake of each lipoprotein by two-thirds. Treatment of liver donor rats with 17 alpha-ethinyl estradiol resulted in a 2-fold increase in the hepatic uptake of VLDL remnants, while cholesterol feeding of liver donor rats caused complete suppression of the receptor-mediated uptake of VLDL remnants. Chylomicron remnant removal was unaffected by estradiol administration and only slightly diminished by cholesterol feeding. The results of competition studies also indicated that a specific chylomicron remnant receptor exists in the liver. Apoprotein E was shown to be required for the receptor-mediated uptake of both remnant lipoproteins. Chylomicron remnants which contained no apoprotein E and VLDL remnants which contained reductively methylated apoprotein E were removed by the liver to about one-third of the extent of native particles. Thus the hepatic uptake of remnant lipoproteins occurs by receptor-mediated processes and the specific removal of both particles is mediated by apoprotein E. In addition, the uptake of VLDL remnants is regulated by the same factors that control hepatic low-density lipoprotein removal, while chylomicron remnant removal is unaffected by these factors.

Removal of lipid-rich lipoproteins by the liver.

Studies were performed to determine the mechanism of hepatic removal of a cholesterol-rich beta-migrating lipoprotein. This fraction, designated IDLc, was isolated from the serum of cholesterol-fed diabetic rats by ultracentrifugation at d 1.006-1.03 g/ml and contained apoproteins B, E, C, and A-I. When 125I-IDLc (125I-labeled IDLc) was injected into normal chow-fed rats, 40% of the radioactivity was cleared from the plasma within 5 minutes with slight additional removal during the next 25 minutes. The rapid removal phase was due to the clearance of apoB-containing lipoproteins. The slow removal phase was due to transfer of apoA-I and C-apoproteins to HDL which has a considerably slower rate of turnover. The in vivo clearance of total 125I-IDLc radioactivity was enhanced by pretreatment of normal rats with 17 alpha-ethinyl estradiol. This appeared to be associated with lack of transfer of apoA-I and C-apoproteins to HDL, and the removal of these apoproteins along with the apoB-containing lipoproteins. Treatment of rats with 17 alpha-ethinyl estradiol did not result in an increased rate of removal of 125I-IDLc when their livers were perfused and this suggests that the removal of IDLc is not mediated by the LDL (B, E) receptor whose activity is stimulated by estradiol administration.

Effects of 17 alpha-ethinyl estradiol on the serum lipoproteins of cholesterol-fed diabetic rats.

Large doses of 17 alpha-ethinyl estradiol were administered to rats made diabetic by administration of streptozotocin and fed a diet containing 2% cholesterol and 1% cholic acid. Before estrogen treatment, these rats were severely hypercholesterolemic and had high concentrations of lipoproteins of d < 1.006 g/ml (very low density lipoproteins, VLDL), d = 1.006 to 1.03 g/ml (intermediate density lipoproteins, IDL), and d = 1.03 to 1.063 g/ml (low density lipoproteins, LDL), and low concentrations of lipoproteins of d = 1.063 to 1.21 g/ml (high density lipoproteins, HDL). The VLDL contained two populations of particles with a mean diameter of 1150 and 350 A, respectively. The IDL contains only the latter particles. These particles are unusual in that they contain apolipoprotein A-I in addition to their usual complement of apolipoproteins. After estrogen treatment, the concentrations of both large and smaller particles in VLDL were greatly decreased as were the concentrations of IDL and LDL; apo A-I was no longer present in the residual VLDL and IDL. HDL phospholipid, protein, and cholesterol increased 7-fold, 4-fold, and 2-fold, respectively. Studies by others have shown that large doses of estrogen enhance uptake of LDL by the liver and this is associated with an increase in the saturable binding sites for LDL on liver membranes. The present studies suggest that these binding sites recognize lipoproteins other than LDL. Furthermore, the enhanced uptake of these lipoproteins appears to result in transfer of their surface components to HDL.

Metabolic alterations associated with the antidiabetic effect of beta 3-adrenergic receptor agonists in obese mice.

Treatment of obese (ob/ob) mice with the beta 3-adrenergic receptor (beta 3-AR) agonist BRL-35135 (1 mg.kg body wt-1.day-1 for 20 days) normalized plasma glucose levels and significantly decreased plasma insulin and nonesterified fatty acid levels. The time frame for the hypoglycemic effect, which reached a maximum after 10 days of treatment, paralleled an increase in brown adipose tissue DNA and protein content. The basal level of mRNA for the beta 3-AR and mitochondrial uncoupling protein was found to be markedly decreased in the ob/ob animals relative to the lean group. Chronic treatment of ob/ob mice for 20 days resulted in a twofold increase in beta 3-AR mRNA and a fivefold increase in uncoupling protein mRNA in brown adipose tissue relative to the placebo group. These findings indicate that chronic treatment of ob/ob animals with a beta 3-AR agonist results in proliferation of brown adipose tissue, with an upregulation of the beta 3-AR, which is associated with a decrease in plasma glucose, insulin, and nonesterified fatty acid levels.

Colloidal gold--low density lipoprotein conjugates as membrane receptor probes.

We have developed a method for conjugating low density lipoproteins (LDL) with colloidal gold. Conjugation, complete after 1 min, occurs by electrostatic adsorption of the LDL to the negatively charged gold particle. Each conjugate consists of approximately eight biologically active LDL molecules clustered around a central 19-nm gold granule. Acidic (pH 4), alkaline (pH 9), or high ionic (600 milliosmolar NaCl) environments do not dissociate the conjugate. Colloidal gold is an electron-dense, nondegradable marker that is easily identified within the cell and serves as a valuable probe for studying receptor binding and endocytosis. By using a modified method of ruthenium red staining, the LDL molecules of the conjugate can be directly visualized when they are bound to the cell surface receptor. Receptor binding (4 degrees C) of the conjugate by cultured human fibroblasts reveals that the gold granule is positioned 18-21 nm from the coated pit region of the membrane. This distance, similar to the diameter of LDL, suggests concomitant internalization of the receptor during vesicular endocytosis and early lysosomal incorporation (10 min at 37 degrees C). Continued internalization (30-60 min at 37 degrees C) results in the formation of free pools of gold within the lysosome.

Ultrastructural visualization of low-density lipoproteins during receptor binding and cellular endocytosis.

Human fibroblasts possess surface receptors which have a high affinity for low-density lipoproteins (LDL). However, previous studies have not provided direct ultrastructural visualization of LDL bound to the receptor. To permit direct observation of unlabeled LDL during receptor binding and cellular endocytosis, we examined several fixative regimens which employ lipophilic stains. Staining with tannic acid, an oxidized form of ruthenium red, or potassium ferrocyanide imparted sufficient contrast to individual molecules of LDL to permit high-resolution electron microscopy of receptor binding and endocytosis. The LDL molecule was observed in immediate contact with the receptor and the coated vesicle, indicating that receptor-ligand binding occurs by short-range interactions.