Rat High Density Lipoprotein Research Articles

Protective effects of aliskiren in doxorubicin-induced acute cardiomyopathy in rats

In this study, effect of aliskiren (ALK) on doxorubicin (DXR)-induced cardiomyopathy in rats was evaluated. ALK (50 and 100 mg/kg/day) was administered for 7 days and a single intraperitoneal injection of DXR (20 mg/kg) on day 5. The animals were sacrificed 48 h after DXR administration. DXR produced significant elevation in malondialdehyde (MDA) and significantly inhibited the activity of glutathione (GSH) in heart tissue, with a significant rise in the serum levels of lactate dehydrogenase (LDH), total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) and reduction in high-density lipoprotein (HDL), indicating acute cardiac toxicity. ALK pretreatment significantly reduced the MDA concentration and ameliorated the inhibition of cardiac GSH activity. ALK also significantly improved the serum levels of LDH, TC, TG, LDL and reduction in HDL in DXR-treated rats. Furthermore, histological examination of the heart sections confirmed the myocardial injury with DXR administration and the near-normal pattern with ALK pretreatment. The results provide clear evidence that the ALK pretreatment offered significant protection against DXR-induced enzymatic changes and cardiac tissue damage.

Lysophosphatidylcholine molecular species in low density lipoprotein and high density lipoprotein in alloxan-induced diabetic rats: effect of probucol.

To elucidate the contribution of diabetic state toward the accumulation of lysophosphatidylcholine (LPC) in low density lipoprotein (LDL), the LPC molecular species in LDL and high density lipoprotein (HDL) obtained from alloxan-induced diabetic rats were determined by high performance liquid chromatography. The palmitoyl LPC (PLPC) per protein was increased in the LDL of diabetic rats (p < 0.05), whereas the stearoyl LPC (SLPC) decreased in both the LDL and HDL of diabetic rats (p < 0.001) in comparison to nondiabetic rats. After the dietary administration of probucol for 4 weeks, the concentrations of SLPC and PLPC in the LDL of diabetic rats and of SLPC in the LDL of nondiabetic rats all significantly decreased by probucol (all; p < 0.01), with a concomitant decrease of the plasma concentrations of total and HDL cholesterol, and phospholipid, compared with those without probucol. The level of TBARS per protein in LDL increased in diabetic rats, and decreased by probucol (p < 0.01). In conclusion, the oxidative stress is thought to be an important factor to accumulate LPC in LDL, although the paradoxical decrease of SLPC in diabetic LDL suggests a non-oxidative metabolism with a preference for the LPC molecular species. The reducing effect of probucol on LPC may not be solely attributed to its antioxidative effect.

Puerarin reduces blood sugar in the diabetic mice and improves hyperlipidemia in rats

The present study was undertaken to observe effects of puerarin (C21H20O9), a component of traditional Chinese drug Gegen (Pueraria root), on hyperlipidemia, blood sugar and sugar tolerance. Methods: 50 male rats were divided into control, hyperlipidemia, hyperlipidemia and Xuezhikang capsule, hyperlipidemia and low dose puerarin, hyperlipidemia and high dose puerarin groups. The hyperlipidemia was induced by high fat diet for 30 days. The levels of serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and HDL/TC were measured. 50 mice were divided into control, diabetes, diabetes and glybenzcyclamide, diabetes and low dose puerarin, diabetes and high dose puerarin groups. The diabetes mice were induced by i.p. alloxan (200mg/kg) for 5 days. Fasting serum sugar, sugar tolerance and insulin receptor gene were measured. The results indicate that puerarin reduced (n=10, P<0.01) levels of serum TG, TC and LDL in rats. Puerarin also increased the level of HDL in rats. It also decreased (n=10, P<0.01) fasting serum sugar in mice and increased (n=10, P<0.01) the sugar tolerance in the diabetic mice. Puerarin increased (n=10, P<0.05) the expression of insulin receptor gene in the diabetic mice. Conclusion: The results suggest that puerarin reduces insulin resistance, and puerarin increasing the expression of insulin receptor gene contributes to its effect of insulin resistance.

Studies on the glycemic and lipidemic effect of monopril and losartan in normal and diabetic rats

The effects of the angiotensin-converting enzyme (ACE) inhibitor monopril and the angiotensin II receptor blocker losartan on serum glucose, protein levels and some serum lipid components were compared in normal and diabetic rats receiving oral antidiabetic drugs ‘repaglinide or gliclazide’. The two antihypertensive agents, when administered concurrently with oral hypoglycemic agents ‘repaglinide or gliclazide’ in normal and diabetic rats exerted a significant hypoglycemic effect. Serum protein levels were mainly unaffected by the two antihypertensive drugs. Monopril and losartan exhibit a hypolipidemic effect in normal and diabetic rats when administered in combination with oral hypoglycemic agents ‘gliclazide or repaglinide’. Monopril or losartan when used alone exerted insignificant effect in high density lipoprotein (HDL) in normal rats, while in combination with gliclazide or repaglinide caused a significant increase in HDL in normal rats. Concomitantly, monopril or losartan, when administered alone or in combination with repaglinide or gliclazide in diabetic rats exerted a significant increase in serum HDL. On the other hand, all the investigated drugs showed a significant decrease in serum low density lipoprotein (LDL) in normal and diabetic rats.

Sterol regulatory element-binding protein-1a binds to cis elements in the promoter of the rat high density lipoprotein receptor SR-BI gene.

The high density lipoprotein (HDL) receptor, or scavenger receptor class B type I (SR-BI), is critical for cholesterol transport and a potential target for hypercholesterolemic drugs. Thus, elucidation of the mechanism underlying regulation of the HDL receptor SR-BI gene is essential. It has been previously shown that there is a correlation between depletion in ovarian cholesteryl ester content and increased HDL receptor SR-BI expression in response to hormonal stimulation. We wanted to determine whether the levels of mature sterol response element-binding protein-1a (SREBP-1a), a key protein in the transcriptional regulation of several genes by sterols, are affected under these conditions. Thus, Western blot analysis was carried out. Consistent with the possibility that SREBP-1a may be involved in the regulation of the HDL receptor SR-BI gene, we found that mature SREBP-1a levels increased up to 11-fold in the ovary after treatment with 50 U hCG. This increase in mature SREBP-1a protein levels correlated with a 30% decrease in ovarian cholesterol levels. These changes in both SREBP-1a and cholesterol levels preceded a 2-fold induction of HDL receptor SR-BI protein levels. To determine whether SREBP-1a could directly regulate the expression of the rat HDL receptor SR-BI gene, approximately 2.2 kb of the receptor SR-BI promoter were cloned and sequenced, and deletion analysis and mobility shift assays were performed. The results of these studies demonstrate that the rat HDL receptor SR-BI promoter contains two sterol response elements (pSRE and dSRE) through which SREBP-1a can bind and activate transcription of this gene. These motifs are similar to known SRE motifs reported for sterol-sensitive genes, and the pSRE is located between two Sp1 sites, similar to the SRE-1 motif in the low density lipoprotein receptor. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, which inhibits SREBP degradation, enhanced the effect of SREBP-1a on the regulation of the rat HDL receptor SR-BI gene. It has previously been shown that tropic hormones such as hCG can also influence gene expression by increasing cAMP levels. Consistent with this fact, we have recently shown that steroidogenic factor-1 (SF-1) mediates cAMP activation of the HDL receptor SR-BI gene. Thus, we decided to examine whether SREBP-1a could cooperate with SF-1 to enhance transcription this gene. The results confirm that indeed both SF-1 and SREBP-1a synergize to induce HDL receptor SR-BI gene expression.

Sterol Regulatory Element-Binding Protein-1a Binds to cis Elements in the Promoter of the Rat High Density Lipoprotein Receptor SR-BI Gene

The high density lipoprotein (HDL) receptor, or scavenger receptor class B type I (SR-BI), is critical for cholesterol transport and a potential target for hypercholesterolemic drugs. Thus, elucidation of the mechanism underlying regulation of the HDL receptor SR-BI gene is essential. It has been previously shown that there is a correlation between depletion in ovarian cholesteryl ester content and increased HDL receptor SR-BI expression in response to hormonal stimulation. We wanted to determine whether the levels of mature sterol response element-binding protein-1a (SREBP-1a), a key protein in the transcriptional regulation of several genes by sterols, are affected under these conditions. Thus, Western blot analysis was carried out. Consistent with the possibility that SREBP-1a may be involved in the regulation of the HDL receptor SR-BI gene, we found that mature SREBP-1a levels increased up to 11-fold in the ovary after treatment with 50 U hCG. This increase in mature SREBP-1a protein levels correlated with a 30% decrease in ovarian cholesterol levels. These changes in both SREBP-1a and cholesterol levels preceded a 2-fold induction of HDL receptor SR-BI protein levels. To determine whether SREBP-1a could directly regulate the expression of the rat HDL receptor SR-BI gene, approximately 2.2 kb of the receptor SR-BI promoter were cloned and sequenced, and deletion analysis and mobility shift assays were performed. The results of these studies demonstrate that the rat HDL receptor SR-BI promoter contains two sterol response elements (pSRE and dSRE) through which SREBP-1a can bind and activate transcription of this gene. These motifs are similar to known SRE motifs reported for sterol-sensitive genes, and the pSRE is located between two Sp1 sites, similar to the SRE-1 motif in the low density lipoprotein receptor. The cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, which inhibits SREBP degradation, enhanced the effect of SREBP-1a on the regulation of the rat HDL receptor SR-BI gene. It has previously been shown that tropic hormones such as hCG can also influence gene expression by increasing cAMP levels. Consistent with this fact, we have recently shown that steroidogenic factor-1 (SF-1) mediates cAMP activation of the HDL receptor SR-BI gene. Thus, we decided to examine whether SREBP-1a could cooperate with SF-1 to enhance transcription this gene. The results confirm that indeed both SF-1 and SREBP-1a synergize to induce HDL receptor SR-BI gene expression.

Steroidogenic factor-1 mediates cyclic 3',5'-adenosine monophosphate regulation of the high density lipoprotein receptor.

The high density lipoprotein (HDL) receptor mediates the uptake of cholesterol and cholesteryl esters, substrates for steroidogenesis, from an HDL particle in the adrenal gland and gonads. We report here that treatment of rat luteal cells with 1 mM (Bu)2cAMP for 24 h dramatically induced (118-fold) HDL receptor messenger RNA levels. The rat HDL receptor promoter contains a steroidogenic factor-1 (SF-1)-binding site (SFBd; 5'-TCAAGGCC-3') through which SF-1 protein binds and activates transcription of this gene in both human HTB9 bladder carcinoma and mouse Y1 tumor cells, an effect that is enhanced by cAMP. These observations demonstrate that this motif is required for both basal and cAMP-induced regulation of the HDL receptor gene. Cotransfection studies in Kin 8 cells, a Y1 cell line resistant to cAMP activation as a result of a mutation in the protein kinase A (PKA) regulatory subunit, showed that a functional PKA is required for cAMP induction of HDL receptor gene transcription. Deleting the activation function-2 domain (amino acids 448-461) or mutating Ser430, a potential consensus phosphorylation site for PKA in the SF-1 protein, decreased both basal and cAMP-induced activation of the HDL receptor promoter. These data suggest that these regions within the SF-1 protein are required for both basal and cAMP-induced regulation of the HDL receptor gene. The mediation of cAMP responsiveness of the HDL receptor gene by SF-1 suggests how important this trans-acting factor is in steroid hormone synthesis by assuring that all required elements (substrate and enzymes) are present when they are needed for maximal steroid production.

Acceleration of development of diabetic cataract by hyperlipidemia and low high-density lipoprotein in rats.

Diabetic cataracts are thought to be caused by hyperglycemia associated with disturbed glucose metabolism. Diabetes mellitus often involves abnormal lipid metabolism in addition to abnormal glucose metabolism. To date, however, very few studies have counted hyperlipidemia as a risk factor for diabetic cataracts. The present study was undertaken to determine whether this abnormal lipid metabolism is a risk factor for diabetic cataracts in rats. Cataracts were caused by streptozotocin (STZ) administration in the ordinary diet or cholesterol rich diet fed rats. When rats with STZ (65 mg/kg)-induced diabetes mellitus were fed an ordinary diet, cataracts became evident at 9 weeks in 26.7% of animals, and increased to an incidence of 53.3% after 10 weeks of STZ treatment. However, in rats with STZ-induced diabetes mellitus that were fed a cholesterol rich diet to induce severe hyperlipidemia and low high density lipoprotein (HDL) cholesterol, cataracts were observed one week earlier, after 8 weeks of treatment, in 40.0% of animals, with an increase to a 53.3% incidence and an 86.7% incidence after 9 and 10 weeks of STZ treatment, respectively. Plasma glucose levels did not differ between the groups. These results suggest that hyperlipidemia and low HDL cholesterol are associated with an earlier onset and an elevated incidence of diabetic cataracts. We then investigated the relationship between plasma lipids and cataracts by STZ (45-85 mg/kg) administration. The results showed that the onset of cataracts correlated positively with plasma total cholesterol, triglyceride, non-HDL cholesterol and glucose levels, and negatively with HDL cholesterol levels. The results of this study suggest that hyperlipidemia and low HDL cholesterol levels may be risk factors for the onset of diabetic cataracts and that diabetic cataracts may be accelerated by hyperlipidemia and low HDL cholesterol in rats.

The Hepatic Uptake of Rat High-Density Lipoprotein Cholesteryl Ester is Delayed After Treatment with Cholesteryl Ester Transfer Protein

The Hepatic Uptake of Rat High-Density Lipoprotein Cholesteryl Ester is Delayed After Treatment with Cholesteryl Ester Transfer Protein

Induction of Adrenal Scavenger Receptor BI and Increased High Density Lipoprotein-Cholesteryl Ether Uptake by in Vivo Inhibition of Hepatic Lipase

Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in cholesterol-utilizing tissues. Inactivation of HL by gene-directed targeting in mice results in up-regulation of SR-BI expression in adrenal gland (Wang, N., Weng, W., Breslow, J. L., and Tall, A. R. (1996) J. Biol. Chem. 271, 21001-21004). The net effect on HDL-cholesteryl ester uptake is not known. We determined the impact of acute in vivo inhibition of rat adrenal HL activity by antibodies on SR-BI expression and on human and rat HDL-[3H]cholesteryl ether (CEth) uptake in the adrenal gland. Rat HDL was isolated from rats in which HL activity had been inhibited for 1 h. The rats were studied under basal conditions (not ACTH-treated) and after previous treatment with ACTH for 6 days (ACTH-treated). Intravenous injection of anti-HL resulted in 70% lowering of adrenal HL activity in both conditions which were maintained for at least 8 h. In not ACTH-treated rats, inhibition of adrenal HL increased adrenal SR-BI mRNA (5.2-fold) and mass (1. 6-fold) within 4 h. HL inhibition resulted in 41% and 14% more adrenal accumulation of human HDL-[3H]CEth during 4 and 24 h, respectively. The adrenal uptake of rat HDL-[3H]CEth increased by 68%, 4 h after the antibody injection. ACTH treatment increased total adrenal HL activity from 3.7 +/- 0.5 milliunits to 34.0 +/- 17. 2 milliunits, as well as adrenal SR-BI mRNA from 2.9 +/- 0.7 arbitrary units (A.U.) to 86.8 +/- 41.1 A.U. and SR-BI mass from 7.7 +/- 1.8 A.U. to 63.16 +/- 46.7 A.U. The human HDL-[3H]CEth uptake by adrenals was also significantly increased from 0.58 +/- 0.11% of injected dose to 7.24 +/- 1.58% of injected dose. Inhibition of adrenal HL activity did not result in further induction of SR-BI expression and did not affect human HDL-[3H]CEth uptake. These findings indicate that SR-BI expression may be influenced by changes in HL activity. HL activity is not needed for the SR-BI-mediated HDL-cholesteryl ester uptake by rat adrenal glands.

Effect of human apolipoprotein A-I gene on vasoactive properties of high density lipoproteins in rats of different age

Vasodilatory activity of high density lipoproteins and the content of apolipoprotein A-I in these particles decreases in senescent rats in comparison with adult animals. Implantation of human apolipoprotein A-I gene increases the content of this apolipoprotein in high density lipoproteins and improves their vasodilatory effect.

Effect of implantation of human apoAI gene on apoprotein composition and vasoactive properties of high-density lipoproteins in rats at different ages

Transfer of human apoAI gene, within the molecular construction which provides its expression, to the liver of adult and aged rats resulted in the appearance of human protein in their blood, and was accompanied by changes in the content of high-density lipoproteins, as well as by the shifts in their protein and lipid composition. Administration of the human ApoAI gene was followed by changes of the vasoactive effects of HDL. Gene implantation is capable of enhancing the direct vasodilatory effects of HDL in old animals, being weakened by ageing, even against the background of normal age changes in the vascular wall tone.

Antibodies against high-density lipoprotein binding proteins enhance high-density lipoprotein uptake but do not affect cholesterol efflux from rat hepatoma cells

High-density lipoprotein plays a key role in the reverse cholesterol transport pathway as well as in the delivery of cholesterol to the liver and steroidogenic tissues. Metabolism of high-density lipoprotein is determined by one of its apolipoproteins, apolipoprotein A-I; however, the identity and function of cellular protein which binds high-density lipoprotein remains unclear. The effect of antibodies against rat high-density lipoprotein binding proteins, HB1 and HB2, on high-density lipoprotein metabolism in a rat hepatoma cell line were studied. Cells were preincubated with the antibodies and 125I-labeled high-density lipoprotein binding and uptake as well as cholesterol biosynthesis and cholesterol efflux to human plasma or isolated high-density lipoprotein were studied. Both antibodies reacted specifically with HB1 and HB2 on the ligand and Western blots, but their binding was not blocked by high-density lipoprotein. Both antibodies inhibited 125I-labeled high-density lipoprotein binding to cells by 20–40%, but stimulated 125I-labeled high-density lipoprotein uptake by up to 2.5-fold. The antibodies had no effect on cholesterol efflux or on cholesterol synthesis. It is concluded that high-density lipoprotein binding proteins, HB1 and HB2, may be involved in high-density lipoprotein uptake in the liver rather than in mediating cholesterol efflux.

Radiolabeled cholesteryl ethers trace LDL cholesteryl esters but not HDL cholesteryl esters in the rat

The intravascular metabolism of cholesteryl [1- 14C]oleoyl ester and [1,2- 3H(N)]cholesteryl palmityl ether was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). The tracers had identical plasma disappearance rates when they were incorporated into human or rat low density lipoproteins (LDL). Fractional catabolic rates (FCR) were 0.081 ± 0.014 h −1 and 0.080 ± 0.013 h −1 for human LDL ester and ether and 0.098 ± 0.007 h −1 and 0.101 ± 0.007 h −1 for rat LDL ester and ether, respectively. In contrast, the ether had plasma disappearance rates that were 24%–25% lower than the ester when they were incorporated into human or rat high density lipoproteins (HDL). FCR were 0.230 ± 0.020 and 0.173 ± 0.030 h −1 for human HDL ester and ether and 0.131 ± 0.020 h −1 and 0.100 ± 0.017 h −1 for rat HDL ester and ether respectively. Biological screening of the rat HDL preparations did not affect these differences. The results of these studies indicate that in the absence of plasma CETA, cholesteryl ethers can be used to trace LDL cholesteryl esters but not to trace HDL cholesteryl esters.

Effect of lipoproteins on cholesterol synthesis in rat Sertoli cells.

Lipoprotein metabolism has been investigated in cultured rat Sertoli cells. Cells incubated with low-density lipoproteins (LDLs) or high-density lipoproteins (HDLs) showed a concentration-dependent decrease of sterol synthesis, indicating a net cholesterol delivery to the Sertoli cells. At 50 micrograms/mL, lipoproteins inhibited the incorporation of [14C]acetate into free cholesterol by 83% for the LDL and 47% for the HDL. Electron microscopic examinations of the Sertoli cells provide evidence of the internalization of gold-labelled HDL into coated pits and coated vesicles. Competitive studies between human LDL and rat HDL indicate that Sertoli cells take up cholesterol from LDL and HDL containing apolipoprotein (apo) E by common pathways. These results suggest that Sertoli cells possess apo B and E receptors for the uptake and degradation of LDL and HDL, although the basement membrane excludes the passage of LDL from blood capillaries to the Sertoli cells. At 50 micrograms/mL, apo-E-depleted HDL inhibited the incorporation of [14C]acetate into free cholesterol by 34%. Thus, this study shows that Sertoli cells are capable of taking up apo-E-depleted HDL cholesterol for cell metabolism.

Comparison of the metabolism of [1,2,6,7- 3H(N)]cholesteryl oleate, cholesteryl [9,10- 3H]oleate, and cholesteryl [1- 14C]oleate labeled lipoproteins in the rat

The intravascular metabolism of sterol labeled [1,2,6,7- 3H(N)]cholesteryl oleate and acyl labeled cholesteryl [9,10- 3H]oleate and cholesteryl [1- 14C]oleate was compared in the rat, an animal species without plasma cholesteryl ester transfer activity (CETA). In a first series of studies, the metabolism of sterol labeled [1,2,6,7- 3H(N)]cholesteryl oleate and acyl labeled cholesteryl [1- 14C]oleate was compared, and the two tracers had identical plasma clearance rates when incorporated into human low density lipoproteins (LDL). The 3H sterol labeled cholesteryl ester (CE), however, had a plasma clearance rate lower than the 14C acyl labeled CE when incorporated into rat a- and β-β-migrating LDL and human or rat high density lipoproteins (HDL). Unesterifed 3H cholesterol reappeared in the plasma whereas the 14C radioactivity in the plasma remained associated with the CE. In a second set of studies, LDL and HDL were radiolabeled with cholesteryl [9,10- 3H]oleate and cholesteryl [1- 14C]oleate Large amounts of 3H radioactivity that were dialyzable and not associated with the lipoprotein CE reappeared in the plasma during the kinetic studies. The two tracers had identical plasma disappearance rates when the plasma samples were dialyzed. The results of these studies indicate that the nature of the tracer used to trace lipoprotein CE can affect the estimated kinetic parameters of plasma CE.

Oyster mushroom ( Pleurotus ostreatus) accelerates the plasma clearance of low-density and high-density lipoproteins in rats

In Wistar rats fed after weaning a semisynthetic diet containing 0.3% cholesterol (C), the addition of 4% dried whole oyster mushroom ( Pleurotus ostreatus) led to a reduced level of serum and liver C at the end of the 10 th week of the experiment by 25, resp. 33%. The level of serum triacylglycerols was not influenced by the mushroom, but was significantly reduced by 13% in liver. The decrease in serum C level is a consequence of the decreased C concentration in very-low-density lipoproteins (VLDL) and in low-density lipoproteins (LDL). Content of C in high-density lipoproteins (HDL) was not influenced by the mushroom. Dietary Pleurotus ostreatus increased the fractional turnover rate of LDL (by 28%) and HDL (by 31%) as determined by the analysis of decay curves of 125I-labelled lipoproteins. The increase in rate of LDL and HDL catabolism is one of the decisive mechanisms which mediates the hypocholesterolemic effect of mushroom in rat.

Identification of high density lipoprotein binding proteins in mature adipocyte plasma membranes.

High density lipoprotein (HDL) binding proteins were identified in nonreduced detergent extracts of plasma membranes or crude membrane fractions of rat adipocytes by ligand blotting. Using 125I-labelled human apolipoprotein-E-free HDL ([125I]HDL3), two binding proteins in adipocyte membranes were detected with apparent molecular masses of 122 and 88 kilodaltons (kDa), respectively. The binding of HDL3 to both binding proteins was abolished by pronase treatment and was inhibited by excess unlabelled HDL3. Excessive unlabelled low density lipoprotein reduced the binding of [125I]HDL3 to the 122-kDa binding protein relatively less than that to the 88-kDa binding protein. Polyclonal antisera against purified rat apolipoprotein A-I (apoA-I) effectively inhibited the binding of HDL3 to adipocyte membranes. Affinity-purified antibodies against rat apoA-I also revealed two HDL-binding proteins in rat adipocyte and liver plasma membranes preincubated with rat HDL. The sizes of the HDL-binding proteins in adipocyte plasma membranes detected by anti-apoA-I were similar to those detected by radiolabelled ligand blotting and their counterparts in rat liver plasma membranes. The study demonstrates two HDL-binding proteins, distinguishable by apparent molecular masses and ligand binding affinity, in plasma membrane proteins of mature rat adipocytes using radiolabelled ligand and immunoligand blotting techniques. The results suggest that apoA-I is involved in the interactions between HDL and both variants of HDL-binding proteins.

Copper deficiency increases hepatic parenchymal cell's maximal binding capacity and impairs Kupffer cell's internalization of apolipoprotein E-free high density lipoprotein in rats

The binding and internalization of apolipoprotein (apo) E-free high density lipoprotein (HDL) by hepatic parenchymal and Kupffer cells were examined with cells and HDL derived from rats fed copper (Cu)-deficient (II nmol/g) and -adequate (126 nmol/g) diets. After 8 weeks of dietary treatment, plasma apo E-free HDL was isolated by a combination of ultracentrifugation, gel filtration, and heparin-Sepharose affinity chromatography. Liver parenchymal and Kupffer cells were obtained by collagenase perfusion and purified by centrifugal elutriation. Freshly isolated cells were incubated with 125I-apo E-free HDL, either from the same treatment group or in a crossover design, to establish if treatment differences were associated with cells, HDL, or both. Binding studies performed at 0° C with increasing apo E-free HDL concentrations demonstrated increases in specific binding and maximum binding capacity (B max) in parenchymal cells from Cu-deficient rats. In addition, cell association studies at 37° C indicated that the amount of apo E-free HDL bound to the cell surface (trypsin releasable) was greater, but the amount internalized (trypsin resistant) was not altered in parenchymal cells from Cu-deficient rats. In contrast, the amount of apo E-free HDL internalized was reduced and that bound to the cell surface was unaltered in Kupffer cells from Cu-deficient rats. Thus Cu deficiency may exert different effects on HDL metabolism in hepatic parenchymal and Kupffer cells. Furthermore, the crossover design demonstrated for the first time that the source of cells from Cu-deficient rats, not HDL, was responsible for the enhanced B max and altered internalization. The results of the present study also support the contention that the hypercholesterolemia associated with the copper-deficient rat model is not the result of decreased HDL uptake by the liver.

Effects of cholesterol uptake from high-density lipoprotein on bile secretion and 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in perfused rat liver

Small aliquots of rat high-density lipoproteins (HDL) (388 ± 67 nmol lipoprotein cholesterol) were labeled with [ 14C]cholesterol and administered as a bolus to perfused rat livers. Bile and perfusate samples were collected for 2 hours at 30-minute intervals. After perfusion, both the microsomes and lipid extracts were prepared from the livers. Lipid composition was examined in both liver and microsomes, and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity was evaluated in microsomes. Basal values of bile flow, lipid composition, and enzyme activity were evaluated using livers in which perfusion was discontinued before injecting the lipoprotein. In some experiments, the effect of perfusion per se was assessed by infusing saline instead of lipoprotein. After 10 minutes of lipoprotein perfusion, 50% of cholesterol administered was taken up by the perfused liver. During infusion, transient but significant increases in both bile flow and bile steroid secretion were observed. Cholesterol administration, even if rapid, represented less than 0.4% of total liver cholesterol content. However, this was enough to significantly increase the cholesterol to phospholipid ( CH PL ) molar ratio in liver microsomes and at the same time decrease HMG-CoA reductase activity. In conclusion, the main response of the perfused liver to HDL cholesterol infusion is a reduced activity of the rate-limiting enzyme in cholesterol biosynthesis, due to the shift in the microsomal CH PL molar ratio. A small proportion of the infused cholesterol enters bile as cholesterol and bile salts.