Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9^−/− mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK¹⁰³↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.     

Targeted Disruption of LDLR Causes Hypercholesterolemia and Atherosclerosis in Yucatan Miniature Pigs

Recent progress in engineering the genomes of large animals has spurred increased interest in developing better animal models for diseases where current options are inadequate. Here, we report the creation of Yucatan miniature pigs with targeted disruptions of the low-density lipoprotein receptor (LDLR) gene in an effort to provide an improved large animal model of familial hypercholesterolemia and atherosclerosis. Yucatan miniature pigs are well established as translational research models because of similarities to humans in physiology, anatomy, genetics, and size. Using recombinant adeno-associated virus-mediated gene targeting and somatic cell nuclear transfer, male and female LDLR+/− pigs were generated. Subsequent breeding of heterozygotes produced LDLR−/− pigs. When fed a standard swine diet (low fat, no cholesterol), LDLR+/− pigs exhibited a moderate, but consistent increase in total and LDL cholesterol, while LDLR−/− pigs had considerably elevated levels. This severe hypercholesterolemia in homozygote animals resulted in atherosclerotic lesions in the coronary arteries and abdominal aorta that resemble human atherosclerosis. These phenotypes were more severe and developed over a shorter time when fed a diet containing natural sources of fat and cholesterol. LDLR-targeted Yucatan miniature pigs offer several advantages over existing large animal models including size, consistency, availability, and versatility. This new model of cardiovascular disease could be an important resource for developing and testing novel detection and treatment strategies for coronary and aortic atherosclerosis and its complications.     

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr^?/?) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE^?/?) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE^?/? mice. In apoE^?/? mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE^?/? mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.     

Control of cholesterol biosynthesis,uptake and storage in hepatocytes by Cideb

Cideb, a member of CIDE family proteins, has emerged as an important regulator in the development of obesity and diabetes by controlling fatty acid synthesis and VLDL secretion in hepatocytes. Here, we investigated the role of Cideb in cholesterol biosynthesis, uptake and storage in the liver by using Cideb-null mice as a model system. Cideb-null mice and wild-type mice were treated with normal diet (ND) or high cholesterol diet (HCD) for one month. The metabolic parameters of cholesterol metabolism and expression profiles of genes in cholesterol biosynthesis and storage were measured. Cideb-null mice had lower levels of plasma cholesterol and LDL when fed with both ND and HCD and increased rate of cholesterol absorption. Furthermore, the liver of Cideb-null mice has lower rates of cholesterol biosynthesis and reduced expression levels of sterol response element-binding protein (SREBP) cleavage-activation protein (SCAP), and lower levels of nuclear form of SREBP2 and its downstream target genes in cholesterol biosynthesis pathway under a normal diet treatment. On the contrary, hepatic cholesterol biosynthesis rate between wild-type and Cideb-null mice was similar after high cholesterol diet treatment. Interestingly, hepatic cholesterol storage in the liver of Cideb-null mice was significantly increased due to its increased LDL receptor (LDLR) and acyl-CoA cholesterol acyltransferase (ACAT) expression. Finally, we observed drastically reduced cholesterol levels in the heart of Cideb-null mice fed with a high cholesterol diet. Overall, our data suggest that Cideb is a novel regulator in controlling cholesterol homeostasis in the liver. Therefore, Cideb could serve as an important therapeutical target for the treatment of atherosclerosis and cardiovascular diseases.     

LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages

Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp ^+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.     

Modulation of lipid homeostasis in response to continuous or intermittent high-fat diet in pigs

A high-fat diet is known to induce atherosclerosis in animal models. Dietary factors and timing of atherogenic food delivery may affect plasma lipoprotein content composition and its potential atherogenic effect. Increasingly often, humans spend periods/days eating in a completely unregulated way, ingesting excessive amounts of food rich in oils and fats, alternating with periods/days when food intake is more or less correct. We investigate the effect on lipid homeostasis of a high-fat diet administered either continuously or intermittently. We investigated control pigs receiving standard diet (C, n=7), pigs receiving a high-fat diet every day for 10 weeks (CHF, n=5), and pigs receiving a high-fat diet every other week for 10 weeks (IHF, n=7). IHF animals were shown to have a different lipid profile compared with CHF animals, with a significant increase in high-density lipoproteins (HDL) levels with respect to C and CHF groups. CHF also showed significantly higher values of TC/HDL cholesterol compared with C and IHF. Hepatic expression analysis of genes involved in lipid homeostasis showed an increasing trend of nuclear receptor LXRα along with its target genes in the CHF group and in the IHF group, whereas SREBP2 and LDLr were significantly inhibited. A significant correlation was found between ABCA1 expression and circulating levels of HDL-C. Periodic withdrawals of a high-fat atherogenic diet compared with a regular administration results in a different adaptive response of lipoprotein metabolism, which leads to a significantly higher plasma level of HDL-C and lower TC/HDL-C.     

TaqIB polymorphism in the CETP gene modulates the impact of HC/LF diet on the HDL profile in healthy Chinese young adults

The aim of this study was to investigate the interactions of genetic variants in the genes of cholesterol ester transfer protein (CETP) and low-density lipoprotein receptor (LDLR) with high carbohydrate and low fat (HC/LF) diet on lipid profiles in a young and healthy Chinese Han population. Fifty-six healthy subjects (22.89±1.80 years) were given washout diets of 31% fat and 54% carbohydrate for 7 days, followed by HC/LF diets of 15% fat and 70% carbohydrate for 6 days, with no total energy restriction. Serum lipid profiles at baseline, after washout and following HC/LF diets, as well as CETP and LDLR polymorphisms were analyzed. Carriers of B2 allele of CETP TaqIB polymorphism had significantly higher levels of high density lipoprotein cholesterol (HDL-C) and apo A-I in the whole study population after the diet intervention. Notably, males with CETP TaqIB B1B1 experienced significantly increased HDL-C and apo A-I after HC/LF diet. Regarding the LDLR Pvu II polymorphism, both P1P1 subjects and P2 carriers experienced decreased total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels after HC/LF diet with no statistically significant differences between the genotypes. Our results demonstrate that the elevated HDL-C levels after HC/LF diet in healthy Chinese Han youth are associated with CETP TaqI B2 allele while males with B1B1 genotype are more susceptible to the influence of HC/LF diet on their HDL-C levels. The decreased TC and LDL-C levels after HC/LF diet are not associated with LDLR Pvu II polymorphism.     

Effect of Artichoke Leaf Extract on Hepatic and Cardiac Oxidative Stress in Rats Fed on High Cholesterol Diet

Hypercholesterolemia and lipid peroxidation play complementary roles in atherosclerosis. Artichoke (Cynara scolymus L., Asteraceae) leaf extract (ALE), rich in antioxidants, has cholesterol-reducing effect. We investigated the effect of ALE on serum and hepatic lipid levels and pro-oxidant–antioxidant balance in the liver and heart of hypercholesterolemic rats. Rats were fed on 4% (w/w) cholesterol and 1% cholic acid (w/w) supplemented diet for 1 month. ALE (1.5 g/kg/day) was given by gavage during the last 2 weeks. High cholesterol (HC) diet caused significant increases in serum and liver cholesterol and triglyceride levels. It increased malondialdehyde (MDA) and diene conjugate (DC) levels in both tissues. Hepatic vitamin E levels and hepatic and cardiac glutathione peroxidase (GSH-Px) activities decreased, but superoxide dismutase and glutathione transferase activities, glutathione, and vitamin C levels remained unchanged due to HC diet. Serum cholesterol and triglyceride levels and ratio of cholesterol to high-density lipoprotein (HDL)-cholesterol decreased in ALE plus HC-treated rats, but liver cholesterol and triglyceride levels remained unchanged. Significant decreases in hepatic and cardiac MDA and DC levels and increases in hepatic vitamin E and GSH-Px activities were observed in ALE-treated hypercholesterolemic rats. Our results indicate that ALE decreases serum lipids and hypercholesterolemia-induced pro-oxidant state in both tissues.     

Modulation of Mannose and Asialoglycoprotein Receptor Expression Determines Glycoprotein Hormone Half-life at Critical Points in the Reproductive Cycle

The rate at which glycoproteins are cleared from the circulation has a critical impact on their biologic activity in vivo. We have shown that clearance rates for glycoproteins such as luteinizing hormone (LH) that undergo regulated release into the circulation determine their potency. Two highly abundant, carbohydrate-specific, endocytic receptors, the asialoglycoprotein receptor (ASGR) and the mannose receptor (ManR) are expressed in the liver by parenchymal and sinusoidal endothelial cells, respectively. We demonstrate that the ManR mediates the clearance of glycoproteins such as LH that bear N-linked glycans terminating with β1,4-linked GalNAc-4-SO₄, as well as glycoproteins bearing glycans that terminate with Man. Steady state levels of mRNA encoding the ASGR and the ManR are regulated by progesterone in pregnant mice, reaching maximal levels on day 12.5 of pregnancy. Protein expression and glycan-specific binding activity also increase in the livers of pregnant mice. In contrast, ManR mRNA, but not ASGR mRNA, decreases in male mice at the time of sexual maturation. We show that levels of ManR and ASGR expression control the clearance rate for glycoproteins bearing recognized glycans. Thus, reduced expression of the ManR at the time of sexual maturation will increase the potency of LH in vivo, whereas increased expression during pregnancy will reduce LH potency until progesterone and receptor levels fall prior to parturition.     

Association between 12α-hydroxylated bile acids and hepatic steatosis in rats fed a high-fat diet

High-fat (HF) diet induces hepatic steatosis that is a risk factor for noncommunicable diseases such as obesity, type 2 diabetes and cardiovascular disease. Previously, we found that HF feeding in rats increases the excretion of fecal bile acids (BAs), specifically 12α-hydroxylated (12αOH) BAs. Although the liver is the metabolic center in our body, the association between hepatic steatosis and 12αOH BAs in HF-fed rats is unclear. Thus, we investigated extensively BA composition in HF-fed rats and evaluated the association between hepatic steatosis and 12αOH BAs. Acclimated male inbred WKAH/HkmSlc rats were divided into two groups and fed either control or HF diet for 8 weeks. Feeding HF diet increased hepatic triglyceride and total cholesterol concentrations, which correlated positively with 12αOH BAs concentrations but not with non-12αOH BAs in the feces, portal plasma and liver. Accompanied by the increase in 12αOH BAs, the rats fed HF diet showed increased fat absorption and higher mRNA expression of liver Cidea. The enhancement of 12αOH BA secretion may contribute to hepatic steatosis by the promotion of dietary fat absorption and hepatic Cidea mRNA expression. The increase in 12αOH BAs was associated with enhanced liver cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) mRNA expression. There was a significant increase in 7α-hydroxycholesterol, a precursor of BAs, in the liver of HF-fed rats. Altogether, these data suggest that the HF diet increases preferentially 12αOH BAs synthesis by utilizing the accumulated hepatic cholesterol and enhancing mRNA expression of Cyp7a1 and Cyp8b1 in the liver.     

Diet-induced lipid accumulation in phospholipid transfer protein-deficient mice: its atherogenicity and potential mechanism

A high saturated fat diet induces free cholesterol and phospholipid accumulation in the plasma of phospholipid transfer protein (Pltp)-deficient mice. In this study, we examined the atherogenic consequence of this phenomenon and investigated the possible mechanism(s). Pltp KO/Apoe KO mice that were fed a coconut oil-enriched high-fat diet (COD) for 7 weeks had higher plasma free cholesterol (149%), phospholipids (15%), and sphingomyelin (54%) than Apoe KO controls. In contrast to chow-fed animals, COD-fed Pltp KO/Apoe KO mice had the same atherosclerotic lesion size as that of Apoe KO mice. Similar to Pltp KO mice, plasma from COD-fed Pltp KO/Apoe KO mice contained VLDL/LDL-sized lamellar particles. Bile measurement indicated that COD-fed Pltp KO mice have 33% less hepatic cholesterol output than controls. In conclusion, COD-fed, Pltp-deficient mice are no longer protected from atherosclerosis and have impaired biliary lipid secretion, which is associated with free cholesterol and phospholipid accumulation.     

Hepatic overexpression of methionine sulfoxide reductase A reduces atherosclerosis in apolipoprotein E-deficient mice

Methionine sulfoxide reductase A (MsrA), a specific enzyme that converts methionine-S-sulfoxide to methionine, plays an important role in the regulation of protein function and the maintenance of redox homeostasis. In this study, we examined the impact of hepatic MsrA overexpression on lipid metabolism and atherosclerosis in apoE-deficient (apoE^−/−) mice. In vitro study showed that in HepG2 cells, lentivirus-mediated human MsrA (hMsrA) overexpression upregulated the expression levels of several key lipoprotein-metabolism-related genes such as liver X receptor α, scavenger receptor class B type I, and ABCA1. ApoE^−/− mice were intravenously injected with lentivirus to achieve high-level hMsrA expression predominantly in the liver. We found that hepatic hMsrA expression significantly reduced plasma VLDL/LDL levels, improved plasma superoxide dismutase, and paraoxonase-1 activities, and decreased plasma serum amyloid A level in apoE^−/− mice fed a Western diet, by significantly altering the expression of several genes in the liver involving cholesterol selective uptake, conversion and excretion into bile, TG biosynthesis, and inflammation. Moreover, overexpression of hMsrA resulted in reduced hepatic steatosis and aortic atherosclerosis. These results suggest that hepatic MsrA may be an effective therapeutic target for ameliorating dyslipidemia and reducing atherosclerosis-related cardiovascular diseases.     

A physiologically based in silico kinetic model predicting plasma cholesterol concentrations in humans

Increased plasma cholesterol concentration is associated with increased risk of cardiovascular disease. This study describes the development, validation, and analysis of a physiologically based kinetic (PBK) model for the prediction of plasma cholesterol concentrations in humans. This model was directly adapted from a PBK model for mice by incorporation of the reaction catalyzed by cholesterol ester transfer protein and contained 21 biochemical reactions and eight different cholesterol pools. The model was calibrated using published data for humans and validated by comparing model predictions on plasma cholesterol levels of subjects with 10 different genetic mutations (including familial hypercholesterolemia and Smith-Lemli-Opitz syndrome) with experimental data. Average model predictions on total cholesterol were accurate within 36% of the experimental data, which was within the experimental margin. Sensitivity analysis of the model indicated that the HDL cholesterol (HDL-C) concentration was mainly dependent on hepatic transport of cholesterol to HDL, cholesterol ester transfer from HDL to non-HDL, and hepatic uptake of cholesterol from non-HDL-C. Thus, the presented PBK model is a valid tool to predict the effect of genetic mutations on cholesterol concentrations, opening the way for future studies on the effect of different drugs on cholesterol levels in various subpopulations in silico.     

Cholesterol homeostasis in guinea pigs fed saturated and polyunsaturated fat diets

Whole body sterol balance, hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity, hepatic low-density lipoprotein (LDL) receptor levels and net tissue cholesterol concentrations were determined in guinea pigs fed either a corn oil- or lard-based purified diet for 6-7 weeks. In comparison to the saturated lard diet, the polyunsaturated corn oil diet resulted in a 34% reduction in plasma total cholesterol levels (P less than 0.02) and a 40% lower triacylglycerol level (P less than 0.02). Feeding the corn oil diet altered very-low-density lipoprotein (VLDL) and LDL composition; the percent cholesterol ester in both particles was decreased and the relative percentages of VLDL triacylglycerol and LDL phospholipid increased. The ratio of surface to core components of LDL from corn oil-fed guinea pigs was significantly higher compared to LDL from animals fed lard. Dietary fat quality had no effect on fecal neutral or acidic steroid excretion, net tissue accumulation of cholesterol, whole body cholesterol synthesis or gallbladder bile composition. Consistent with these results was the finding that fat quality did not alter either expressed (non-phosphorylated) or total hepatic HMG-CoA reductase activities. The hepatic concentrations of free and esterified cholesterol were significantly increased in corn oil-fed animals, as were cholesterol concentrations in intestine, adipose tissue, muscle and total carcass. Analysis of receptor-mediated LDL binding to isolated hepatic membranes demonstrated that the polyunsaturated corn-oil based diet caused a 1.9-fold increase in receptor levels (P less than 0.02). The data indicate that the hypocholesterolemic effects of dietary polyunsaturated fat in the guinea pig are not attributable to changes in endogenous cholesterol synthesis or catabolism but rather may result from a redistribution of plasma cholesterol to body tissue due to an increase in tissue LDL receptors.