A novel mechanism by which probucol lowers low density lipoprotein levels demonstrated in the LDL receptor-deficient rabbit

Treatment of low density lipoprotein (LDL) receptor-deficient rabbits (WHHL rabbits) with probucol (1% w/w in a chow diet) lowered their LDL-cholesterol levels by 36%, consonant with the reported effectiveness of the drug in patients deficient in the LDL receptor. Initial studies of LDL fractional catabolic rate (FCR) using 125I-labeled LDL prepared from the serum of untreated WHHL rabbits showed no difference between probucol-treated WHHL rabbits and untreated WHHL rabbits. When, however, 125I-labeled LDL was prepared from donor WHHL rabbits under treatment with probucol and injected back into them, the FCR was found to be increased by about 50% above that measured simultaneously using 131I-labeled LDL prepared from untreated WHHL donors. The labeled LDL from probucol-treated donors was also metabolized more rapidly than that from untreated donors when injected into untreated WHHL rabbits or into untreated wild-type New Zealand White rabbits. Finally, it was shown that rabbit skin fibroblasts in culture degraded labeled LDL prepared from probucol-treated WHHL rabbits more rapidly than that prepared from untreated WHHL donors. This was true both for normal rabbit fibroblasts and also for WHHL skin fibroblasts, although the absolute degradation rates in the latter were, of course, much lower for both forms of LDL. The data indicate that a major mechanism by which probucol lowers LDL levels relates not to changes in the cellular mechanisms for LDL uptake or to changes in LDL production but rather to intrinsic changes in the structure and metabolism of the plasma LDL of the probucol-treated animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice

To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL-WT) with mice expressing inactive HL (HL-S145G) in a background lacking endogenous HL and the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (P < 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous (125)I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day(-1): 6.07 +/- 0.25, LDLr-KOxHL-WT; 4.76 +/- 0.30, LDLr-KOxHL-S145G; 3.70 +/- 0.13, LDLr-KOxHL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KOxHL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P < 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice.     

Protection against atherosclerosis by estrogen is independent of plasma cholesterol levels in LDL receptor-deficient mice

Low density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice consuming a high fat diet were used to assess the effect of endogenous and exogenous estradiol (E2) on atherosclerosis. Sexually mature female mice were ovariectomized (OVX) and implanted with subcutaneous, slow-release pellets designed to release 6 microg/day of exogenous 17beta-estradiol (17beta-E2 ), 17alpha-estradiol (17alpha-E2 ), or placebo (E2- deficient). Sham-operated control female (endogenous E2 ) and male mice were studied as controls. Aortic atherosclerotic lesion area was reduced by physiologic amounts of both endogenous and exogenous E2 compared to E2-deficient female mice. Although plasma cholesterol levels were reduced by exogenous E2 despite the absence of the LDL receptor, endogenous E2 was not associated with any cholesterol changes. In contrast, only 17alpha-E2 was associated with decreased fasting triglyceride. In subgroup analyses matched for time-averaged plasma total cholesterol, aortic lesion area was reduced by the presence of estradiol (E2 ). E2 protected LDLR-/- female mice from atherosclerosis and this protection was independent of changes in plasma cholesterol levels.     

Hyperlipidemia is a major determinant of neointimal formation in LDL receptor-deficient mice

LDL receptor-deficient (LDLR(-/-)) mice exhibit mild hyperlipidemia on a chow diet but develop severe hyperlipidemia on a high fat diet. In this study, we investigated neointimal formation after removal of the endothelium when LDLR(-/-) mice were fed chow or a Western diet containing 42% fat, 0.15% cholesterol, and 19.5% casein. At 10 weeks of age, female mice underwent endothelial denudation of the left common carotid artery. Two weeks after injury, neointimal formation was barely detectable in the injured vessel when mice developed mild hyperlipidemia on the chow diet. In contrast, neointimal lesions were obvious when mice developed severe hyperlipidemia on the Western diet. Immunohistochemical and histological analyses demonstrated the presence of macrophage foam cells and smooth muscle cells in neointimal lesions. The injured artery also exhibited a significant increase in medial area on the Western diet. Plasma levels of MCP-1 and soluble VCAM-1 were significantly elevated by feeding of the Western diet. These data indicate that hyperlipidemia aggravates neointimal growth in LDLR(-/-) mice by promoting foam cell formation and inflammation.     

Antisense oligonucleotide reduction of apoB-ameliorated atherosclerosis in LDL receptor-deficient mice

Chronic elevations of plasma apolipoprotein B (apoB) are strongly associated with cardiovascular disease. We have previously demonstrated that inhibition of hepatic apoB mRNA using antisense oligonucleotides (ASO) results in reductions of apoB, VLDL, and LDL in several preclinical animal models and humans. In this study, we evaluated the anti-atherogenic effects of a murine-specific apoB ASO (ISIS 147764) in hypercholesterolemic LDLr deficient (LDLr(-/-)) mice. ISIS 147764 was administered weekly at 25-100 mg/kg for 10-12 weeks and produced dose-dependent reductions of hepatic apoB mRNA and plasma LDL by 60-90%. No effects on these parameters were seen in mice receiving control ASOs. ApoB ASO treatment also produced dose-dependent reductions of aortic en face and sinus atherosclerosis from 50-90%, with high-dose treatment displaying less disease than the saline-treated, chow-fed LDLr(-/-) mice. No changes in intestinal cholesterol absorption were seen with apoB ASO treatment, suggesting that the cholesterol-lowering pharmacology of 147764 was primarily due to inhibition of hepatic apoB synthesis and secretion. In summary, ASO-mediated suppression of apoB mRNA expression profoundly reduced plasma lipids and atherogenesis in LDLr(-/-) mice, leading to the hypothesis that apoB inhibition in humans with impaired LDLr activity may produce similar effects.     

Growth hormone reduces plasma cholesterol in LDL receptor-deficient mice.

Growth hormone (GH) has pleiotropic effects on cholesterol and lipoprotein metabolism. Pituitary GH is important for the normal regulation of hepatic LDL receptors (LDLR), for the enzymatic activity of bile acid regulatory cholesterol 7alpha-hydroxylase (C7alphaOH), and for the maintenance of resistance to dietary cholesterol. The present study aimed to determine whether GH has beneficial effects on plasma lipids and hepatic cholesterol metabolism in mice devoid of LDLR. Compared with wild-type controls, LDLR-deficient mice had approximately 250% elevated plasma total cholesterol and approximately 50% increased hepatic cholesterol levels; hepatic HMG CoA reductase activity was reduced by 70%, whereas C7alphaOH activity was increased by 40%. In LDLR mice, GH infusion reduced plasma cholesterol and triglycerides up to 40%, whereas HMG CoA reductase and C7alphaOH activities were stimulated by approximately 50% and 110% respectively. GH also stimulated HMG CoA reductase and C7alphaOH activities in control mice, whereas hepatic LDLR and plasma lipoproteins were unchanged. The effects of cholestyramine and atorvastatin on C7alphaOH in LDLR-deficient mice were potentiated by GH, and this was associated with a further reduction in plasma cholesterol. GH treatment reduces plasma cholesterol and triglycerides and stimulates C7alphaOH activity in mice devoid of LDLR, particularly in combination with resin or statin treatment. The potential of GH therapy in patients with homozygous familial hypercholesterolemia should be evaluated.     

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Effects of coexpression of the LDL receptor and apoE on cholesterol metabolism and atherosclerosis in LDL receptor-deficient mice

The low density lipoprotein receptor (LDLR) plays a major role in regulation of plasma cholesterol levels as a ligand for apolipoprotein B-100 and apolipoprotein E (apoE). Consequently, LDLR-deficient mice fed a Western-type diet develop significant hypercholesterolemia and atherosclerosis. ApoE not only mediates uptake of atherogenic lipoproteins via the LDLR and other cell-surface receptors, but also directly inhibits atherosclerosis. In this study, we examined the hypothesis that coexpression of the LDLR and apoE would have greater effects than either one alone on plasma cholesterol levels and the development of atherosclerosis in LDLR-deficient mice. LDLR-deficient mice fed a Western-type diet for 10 weeks were injected with recombinant adenoviral vectors encoding the genes for human LDLR, human apoE3, both LDLR and apoE3, or lacZ (control). Plasma lipids were analyzed at several time points after vector injection. Six weeks after injection, mice were analyzed for extent of atherosclerosis by two independent methods. As expected, LDLR expression alone induced a significant reduction in plasma cholesterol due to reduced VLDL and LDL cholesterol levels, whereas overexpression of apoE alone did not reduce plasma cholesterol levels. When the LDLR and apoE were coexpressed in this model, the effects on plasma cholesterol levels were no greater than with expression of the LDLR alone. However, coexpression did result in a substantial increase in large apoE-rich HDL particles. In addition, although the combination of cholesterol reduction and apoE expression significantly reduced atherosclerosis, its effects were no greater than with expression of the LDLR or apoE alone. In summary, in this LDLR-deficient mouse model fed a Western-type diet, there was no evidence of an additive effect of expression of the LDLR and apoE on cholesterol reduction or atherosclerosis.     

Low density lipoprotein (LDL)-mediated suppression of Lewis lung carcinoma in hypercholesterolemic LDL receptor-deficient mice

An inverse relationship has been reported between cancer risk and cholesterol level, prompting the hypothesis that hypercholesterolemia may be protective against cancer. We tested this hypothesis by evaluating the growth of Lewis lung carcinoma in three different murine models of hypercholesterolemia: Pluronic treated mice, apolipoprotein E (ApoE) deficient mice, and low density lipoprotein receptor (LDL-R) deficient mice. Only the accumulation of LDL-cholesterol in LDL-R deficient mice suppressed tumor growth. Accumulation of chylomicrons, very low density lipoproteins (VLDL), and cholesterol-enriched remnants in the Pluronic treated mice and ApoE deficient mice did not inhibit tumor growth, even though mice in all three models were equally hypercholesterolemic. Taken together, the experimental evidence from our studies indicate that high plasma cholesterol in the form of LDL-cholesterol could have a beneficial effect against cancer in vivo.     

The bridging function of hepatic lipase clears plasma cholesterol in LDL receptor-deficient "apoB-48-only" and "apoB-100-only" mice

Hepatic lipase clears plasma cholesterol by lipolytic and nonlipolytic processing of lipoproteins. We hypothesized that the nonlipolytic processing (known as the bridging function) clears cholesterol by removing apoB-48- and apoB-100-containing lipoproteins by whole particle uptake. To test our hypotheses, we expressed catalytically inactive human HL (ciHL) in LDL receptor deficient "apoB-48-only" and "apoB-100-only" mice. Expression of ciHL in "apoB-48-only" mice reduced cholesterol by reducing LDL-C (by 54%, 46 +/- 6 vs. 19 +/- 8 mg/dl, P < 0.001). ApoB-48 was similarly reduced (by 60%). The similar reductions in LDL-C and apoB-48 indicate cholesterol removal by whole particle uptake. Expression of ciHL in "apoB-100-only" mice reduced cholesterol by reducing IDL-C (by 37%, 61 +/- 19 vs. 38 +/- 12 mg/dl, P < 0.003). Apo-B100 was also reduced (by 27%). The contribution of nutritional influences was examined with a high-fat diet challenge in the "apoB-100-only" background. On the high fat diet, ciHL reduced IDL-C (by 30%, 355 +/- 72 vs. 257 +/- 64 mg/dl, P < 0.04) but did not reduce apoB-100. The reduction in IDL-C in excess of apoB-100 suggests removal either by selective cholesteryl ester uptake, or by selective removal of larger, cholesteryl ester-enriched particles. Our results demonstrate that the bridging function removes apoB-48- and apoB-100-containing lipoproteins by whole particle uptake and other mechanisms.     

Transgenic expression of CYP7A1 in LDL receptor-deficient mice blocks diet-induced hypercholesterolemia

Constitutive expression of a cholesterol-7alpha-hydroxylase (CYP7A1) transgene in LDL receptor-deficient mice blocked the ability of a cholesterol-enriched diet to increase plasma levels of apolipoprotein B-containing lipoproteins. LDL receptor-deficient mice expressing the CYP7A1 transgene exhibited complete resistance to diet-induced hypercholesterolemia and to the accumulation of cholesterol in the liver. Hepatic mRNA expression of liver X receptor-inducible ABCG5 and ABCG8 was decreased in CYP7A1 transgenic, LDL receptor-deficient mice fed a cholesterol-enriched diet. Thus, increased biliary cholesterol excretion could not account for the maintenance of cholesterol homeostasis. CYP7A1 transgenic, LDL receptor-deficient mice fed the cholesterol-enriched diet exhibited decreased jejunal Niemann-Pick C1-Like 1 protein (NPC1L1) mRNA expression, an important mediator of intestinal cholesterol absorption. A taurocholate-enriched diet also decreased NPC1L1 mRNA expression in a farnesoid X receptor-independent manner. Reduced expression of NPC1L1 mRNA was associated with decreased cholesterol absorption ( approximately 20%; P < 0.05) exhibited by CYP7A1 transgenic LDL receptor-deficient mice fed the cholesterol-enriched diet. The combined data show that enhanced expression of CYP7A1 is an effective means to prevent the accumulation of cholesterol in the liver and of atherogenic apolipoprotein B-containing lipoproteins in plasma.     

PANDORA-Seq unveils the hidden small noncoding RNA landscape in atherosclerosis of LDL receptor-deficient mice

Small noncoding RNAs (sncRNAs) play diverse roles in numerous biological processes. While the widely used RNA sequencing (RNA-Seq) method has advanced sncRNA discovery, RNA modifications can interfere with the complementary DNA library construction process, preventing the discovery of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-Seq (Panoramic RNA Display by Overcoming RNA Modification Aborted Sequencing) method to overcome RNA modification-elicited sequence interferences. To identify novel sncRNAs associated with atherosclerosis development, LDL receptor-deficient (LDLR^−/−) mice were fed a low-cholesterol diet or high-cholesterol diet (HCD) for 9 weeks. Total RNAs isolated from the intima were subjected to PANDORA-Seq and traditional RNA-Seq. By overcoming RNA modification-elicited limitations, PANDORA-Seq unveiled an rsRNA/tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR^−/− mice, which was strikingly different from that detected by traditional RNA-Seq. While microRNAs were the dominant sncRNAs detected by traditional RNA-Seq, PANDORA-Seq substantially increased the reads of rsRNAs and tsRNAs. PANDORA-Seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding, including 1,160 rsRNAs and 195 tsRNAs. One of HCD-induced intimal tsRNAs, tsRNA-Arg-CCG, may contribute to atherosclerosis development by regulating the proatherogenic gene expression in endothelial cells. Overall, PANDORA-Seq revealed a hidden rsRNA and tsRNA population associated with atherosclerosis development. These understudied tsRNAs and rsRNAs, which are much more abundant than microRNAs in the atherosclerotic intima of LDLR^−/− mice, warrant further investigations.     

LDL receptor deficiency or apoE mutations prevent remnant clearance and induce hypertriglyceridemia in mice

We have used adenovirus-mediated gene transfer and bolus injection of purified apolipoprotein E (apoE) in mice to determine the contribution of LDL receptor family members in the clearance of apoE-containing lipoproteins in vivo and the factors that trigger hypertriglyceridemia. A low dose [5 x 10(8) plaque-forming units (pfu)] of an adenovirus expressing apoE4 did not normalize plasma cholesterol levels of apolipoprotein E-deficient (apoE(-/-)) x low density lipoprotein receptor-deficient (LDLr(-/-)) mice and induced hypertriglyceridemia. A similar phenotype of combined dyslipidemia was induced in apoE(-/-) or apoE(-/-) x LDLr(-/-) mice after infection with a low dose (4 x 10(8) pfu) of an adenovirus expressing the apoE4[R142V/R145V] mutant previously shown to be defective in receptor binding. In contrast, a low dose of 5 x 10(8) pfu of the apoE4-expressing adenovirus corrected hypercholesterolemia in apoE(-/-) mice and did not trigger hypertriglyceridemia. Bolus injection of purified apoE in apoE(-/-) x LDLr(-/-) mice did not clear plasma cholesterol levels and induced mild hypertriglyceridemia. In contrast, similar injection of apoE in apoE(-/-) mice cleared plasma cholesterol and caused transiently mild hypertriglyceridemia. These findings suggest that a) the LDL receptor alone can account for the clearance of apoE-containing lipoproteins in mice, and the contribution of other receptors is minimal, and b) defects in either the LDL receptor or in apoE that affect its interactions with the LDL receptor, increase the sensitivity to apoE-induced hypertriglyceridemia in mice.     

T-0901317, a synthetic liver X receptor ligand,inhibits development of atherosclerosis in LDL receptor-deficient mice

Liver X receptors (LXR alpha and LXR beta) are nuclear receptors, which are important regulators of cholesterol and lipid metabolism. LXRs control genes involved in cholesterol efflux in macrophages, bile acid synthesis in liver and intestinal cholesterol absorption. LXRs also regulate genes participating in lipogenesis. To determine whether the activation of LXR promotes or inhibits development of atherosclerosis, T-0901317, a synthetic LXR ligand, was administered to low density lipoprotein receptor (LDLR)(-/-) mice. T-0901317 significantly reduced the atherosclerotic lesions in LDLR(-/-) mice without affecting plasma total cholesterol levels. This anti-atherogenic effect correlated with the plasma concentration of T-0901317, but not with high density lipoprotein cholesterol, which was increased by T-0901317. In addition, we observed that T-0901317 increased expression of ATP binding cassette A1 in the lesions in LDLR(-/-) mice as well as in mouse peritoneal macrophages. T-0901317 also significantly induced cholesterol efflux activity in peritoneal macrophages. These results suggest that LXR ligands may be useful therapeutic agents for the treatment of atherosclerosis.     

Elaiophylin reduces body weight and lowers glucose levels in obese mice by activating AMPK

Obesity is an epidemic affecting 13% of the global population and increasing the risk of many chronic diseases. However, only several drugs are licensed for pharmacological intervention for the treatment of obesity. As a master regulator of metabolism, the therapeutic potential of AMPK is widely recognized and aggressively pursued for the treatment of metabolic diseases. We found that elaiophylin (Ela) rapidly activates AMPK in a panel of cancer-cell lines, as well as primary hepatocytes and adipocytes. Meanwhile, Ela inhibits the mTORC1 complex, turning on catabolism and turning off anabolism together with AMPK. In vitro and in vivo studies showed that Ela does not activate AMPK directly, instead, it increases cellular AMP/ATP and ADP/ATP ratios, leading to AMPK phosphorylation in a LKB1-dependent manner. AMPK activation induced by Ela caused changes in diverse metabolic genes, thereby promoting glucose consumption and fatty acid oxidation. Importantly, Ela activates AMPK in mouse liver and adipose tissue. As a consequence, it reduces body weight and blood glucose levels and improves glucose and insulin tolerance in both ob/ob and high-fat diet-induced obese mouse models. Our study has identified a novel AMPK activator as a candidate drug for the treatment of obesity and its associated chronic diseases.Subject terms: Cell biology, Drug discovery     

Macrophage lipoprotein lipase promotes foam cell formation and atherosclerosis in low density lipoprotein receptor-deficient mice

The role of macrophage lipoprotein lipase (LPL) expression in atherosclerotic lesion formation was examined in low density lipoprotein receptor (LDLR(-/-)) mice using dietary conditions designed to induce either fatty streak lesions or complex atherosclerotic lesions. First, LDLR(-/-) mice chimeric for macrophage LPL expression were created by transplantation of lethally irradiated female LDLR(-/-) mice with LPL(-/-) (n = 12) or LPL(+/+) (n = 14) fetal liver cells as a source of hematopoietic cells. To induce fatty streak lesions, these mice were fed a Western diet for 8 weeks, resulting in severe hypercholesterolemia. There were no differences in plasma post-heparin LPL activity, serum lipid levels, or lipoprotein distribution between these two groups. The mean lesion area in the proximal aorta in LPL(-/-) --> LDLR(-/-) mice was significantly reduced by 33% compared with LPL(+/+) --> LDLR(-/-) mice, and a similar reduction (38%) in lesion area was found by en face analysis of the aortae. To induce complex atherosclerotic lesions, female LDLR(-/-) mice were lethally irradiated, transplanted with LPL(-/-) (n = 14), LPL(+/-) (n = 13), or LPL(+/+) (n = 14) fetal liver cells, and fed the Western diet for 19 weeks. Serum cholesterol and triglyceride levels did not differ between the three groups. After 19 weeks of diet, the lesions in the proximal aorta were complex with relatively few macrophages expressing LPL protein and mRNA in LPL(+/+) --> LDLR(-/-) mice. Analysis of cross-sections of the proximal aorta demonstrated no differences in the extent of lesion area between the groups, whereas en face analysis of the aortae revealed a dose-dependent effect of macrophage LPL on mean aortic lesion area in LPL(-/-) --> LDLR(-/-), LPL(-/+) --> LDLR(-/-), and LPL(+/+) --> LDLR(-/-) mice (1.8 +/- 0. 2%, 3.5 +/- 0.5% and 5.9 +/- 0.8%, respectively). Taken together, these data indicate that macrophage LPL expression in the artery wall promotes atherogenesis during foam cell lesion formation, but this impact may be limited to macrophage-rich lesions.     

Catalytically inactive lipoprotein lipase overexpression increases insulin sensitivity in mice.

Abnormalities in lipoprotein lipase (LPL) function contribute to the development of hypertriglyceridemia, one of the characteristic disorders observed in the metabolic syndrome. In addition to the hydrolyzing activity of triglycerides, LPL modulates various cellular functions via its binding ability to the cell surface. Here we show the effects of catalytically inactive LPL overexpression on high-fat diet (HFD)-induced decreased systemic insulin sensitivity in mice. The binding capacity of catalytically inactive G188E-LPL to C2C12 skeletal muscle cells was not significantly different from that of wild type LPL. Insulin-stimulated IRS-1 phosphorylation and glucose uptake were increased by addition of wild type or mutant LPL in C2C12 cells. After 10 weeks' of HFD feeding, mice had significantly higher blood glucose levels than chow-fed mice in insulin tolerance tests. The blood glucose levels after insulin injection was significantly decreased in mutated LPL-overexpressing mice (G188E mice), as well as in wild type LPL-overexpressing mice (WT mice). Overexpression of catalytically inactive LPL, as well as wild type LPL, improved impaired insulin sensitivity in mice. These results show that decreased expression of LPL possibly causes the insulin resistance, in addition to hypertriglyceridemia, in metabolic syndrome.     

Effects of emulsified policosanols with different chain lengths on cholesterol metabolism in heterozygous LDL receptor-deficient mice

Policosanol is a mixture of long-chain primary aliphatic saturated alcohols. Previous studies in humans and animals have shown that these compounds improved lipoprotein profiles. However, more-recent placebo-controlled studies could not confirm these promising effects. Octacosanol (C28), the main component of sugarcane-derived policosanol, is assumed to be the bioactive component. This has, however, never been tested in an in vivo study that compared individual policosanol components side by side. Here we present that neither the individual policosanol components (C24, C26, C28, or C30) nor the natural policosanol mixture (all 30 mg/100 g diet) lowered serum cholesterol concentrations in LDL receptor knock-out (LDLr(+/-)) mice. Moreover, there was no effect on gene expression profiles of LDLr, ABCA1, HMG-CoA synthase 1, and apolipoprotein A-I (apoA-I) in hepatic and small intestinal tissue of female LDLr(+/-) mice after the 7 week intervention period. Finally, none of the individual policosanols or their respective long-chain fatty acids or aldehydes affected de novo apoA-I protein production in vitro in HepG2 and CaCo-2 cells. Therefore, we conclude that the evaluated individual policosanols, as well as the natural policosanol mixture, have no potential for reducing coronary heart disease risk through effects on serum lipoprotein concentrations.     

An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis

High levels of plasma apolipoprotein B-100 (apoB-100), the principal apolipoprotein of LDL, are associated with cardiovascular disease. We hypothesized that suppression of apoB-100 mRNA by an antisense oligonucleotide (ASO) would reduce LDL cholesterol (LDL-C). Because most of the plasma apoB is made in the liver, and antisense drugs distribute to that organ, we tested the effects of a mouse-specific apoB-100 ASO in several mouse models of hyperlipidemia, including C57BL/6 mice fed a high-fat diet, Apoe-deficient mice, and Ldlr-deficient mice. The lead apoB-100 antisense compound, ISIS 147764, reduced apoB-100 mRNA levels in the liver and serum apoB-100 levels in a dose- and time-dependent manner. Consistent with those findings, total cholesterol and LDL-C decreased by 25-55% and 40-88%, respectively. Unlike small-molecule inhibitors of microsomal triglyceride transfer protein, ISIS 147764 did not produce hepatic or intestinal steatosis and did not affect dietary fat absorption or elevate plasma transaminase levels. These findings, as well as those derived from interim phase I data with a human apoB-100 antisense drug, suggest that antisense inhibition of this target may be a safe and effective approach for the treatment of humans with hyperlipidemia.