Inhibition of ASGR1 decreases lipid levels by promoting cholesterol excretion.

High cholesterol is a major risk factor for cardiovascular disease1. Currently, no drug lowers cholesterol through directly promoting cholesterol excretion. Human genetic studies have identified that the loss-of-function Asialoglycoprotein receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of cardiovascular disease2. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins3. The mechanism by which ASGR1 affects cholesterol metabolism is unknown. Here, we find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXRα. LXRα upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein and excretion to bile and faeces4, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino-acid levels in lysosomes, and thereby inhibits mTORC1 and activates AMPK. On one hand, AMPK increases LXRα by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolaemic drugs. In summary, this study demonstrates that targeting ASGR1 upregulates LXRα, ABCA1 and ABCG5/G8, inhibits SREBP1 and lipogenesis, and therefore promotes cholesterol excretion and decreases lipid levels.

Silencing Porcine CMAH and GGTA1 Genes Significantly Reduces Xenogeneic Consumption of Human Platelets by Porcine Livers.

BACKGROUND: A profound thrombocytopenia limits hepatic xenotransplantation in the pig-to-primate model. Porcine livers also have shown the ability to phagocytose human platelets in the absence of immune-mediated injury. Recently, inactivation of the porcine ASGR1 gene has been shown to decrease this phenomenon. Inactivating GGTA1 and CMAH genes has reduced the antibody-mediated barrier to xenotransplantation; herein, we describe the effect that these modifications have on xenogeneic consumption of human platelets in the absence of immune-mediated graft injury. METHODS: Wild type (WT), ASGR1, GGTA1, and GGTA1CMAH knockout pigs were compared for their xenogeneic hepatic consumption of human platelets. An in vitro assay was established to measure the association of human platelets with liver sinusoidal endothelial cells (LSECs) by immunohistochemistry. Perfusion models were used to measure human platelet uptake in livers from WT, ASGR1, GGTA1, and GGTA1 CMAH pigs. RESULTS: GGTA1, CMAH LSECs exhibited reduced levels of human platelet binding in vitro when compared with GGTA1 and WT LSECs. In a continuous perfusion model, GGTA1 CMAH livers consumed fewer human platelets than GGTA1 and WT livers. GGTA1 CMAH livers also consumed fewer human platelets than ASGR1 livers in a single-pass model. CONCLUSIONS: Silencing the porcine carbohydrate genes necessary to avoid antibody-mediated rejection in a pig-to-human model also reduces the xenogeneic consumption of human platelets by the porcine liver. The combination of these genetic modifications may be an effective strategy to limit the thrombocytopenia associated with pig-to-human hepatic xenotransplantation.

Susceptibility to T cell-mediated liver injury is enhanced in asialoglycoprotein receptor-deficient mice.

T cell activation and associated pro-inflammatory cytokine production is a pathological feature of inflammatory liver disease. It is also known that liver injury is associated with marked impairments in the function of many hepatic proteins including a hepatocyte-specific binding protein, the asialoglycoprotein receptor (ASGPR). Recently, it has been suggested that hepatic ASGPRs may play an important role in the physiological regulation of T lymphocytes, leading to our hypothesis that ASGPR defects correlate with inflammatory-mediated events in liver diseases. Therefore, in this study we investigated whether changes in hepatocellular ASGPR expression were related to the dysregulation of intrahepatic T lymphocytes and correlate with the development of T-cell mediated hepatitis. Mice lacking functional ASGPRs (receptor-deficient, RD), and wild-type (WT) controls were intravenously injected with T-cell mitogens, Concanavalin A (Con A) or anti-CD3 antibody. As a result of T cell mitogen treatment, RD mice lacking hepatic ASGPRs displayed enhancements in liver pathology, transaminase activities, proinflammatory cytokine expression, and caspase activation compared to that observed in normal WT mice. Furthermore, FACS analysis demonstrated that T-cell mitogen administration resulted in a significant rise in the percentage of CD8+ lymphocytes present in the livers of RD animals versus WT mice. Since these two mouse strains differ only in whether they express the hepatic ASGPR, it can be concluded that proper ASGPR function exerts a protective effect against T cell mediated hepatitis and that impairments to this hepatic receptor could be related to the accumulation of cytotoxic T cells that are observed in inflammatory liver diseases.

Impact of asialoglycoprotein receptor deficiency on the development of liver injury.

The asialoglycoprotein (ASGP) receptor is a well-characterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis (RME). The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the mis-sorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously, we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease (ALD). The studies revealed profound ethanol-mediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations, studies were performed utilizing knockout mice (lacking a functional ASGP receptor) to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti-Fas (CD95) antibody, carbon tetrachloride (CCl(4)) and lipopolysaccharide (LPS)/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels, histopathological scores, as well as hepatocellular death. Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxin-mediated liver diseases.

Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice.

The asialoglycoprotein (ASGP) receptor is an abundant hepatocyte-specific receptor involved in receptor-mediated endocytosis. This receptor's abundance and function is decreased by chronic ethanol administration prior to the appearance of pathology such as necrosis or inflammation. Hence, this study aimed to determine if ASGP receptor function is required to protect against liver injury by utilizing a knockout mouse model lacking functional ASGP receptor in the setting of carbon tetrachloride (CCl(4)) hepatotoxicity. Briefly, ASGP receptor-deficient (RD) mice and wild-type (WT) mice were injected with 1ml/kg body weight of CCl(4). In the subsequent week, mice were monitored for liver damage and pathology (aspartate transaminase (AST), alanine transaminase (ALT) and light microscopy). The consequences of CCl(4) injection were examined by measuring alpha-smooth muscle actin (alpha-SMA) deposition, contents of malondialdehyde and the percentage of apoptotic hepatocytes. After CCl(4) injection, RD mice showed increased liver pathology together with significantly increased activities of AST and ALT compared to that in WT mice. There were also significantly more apoptotic bodies, lipid peroxidation and deposition of alpha-SMA in RD mice versus WT mice following CCl(4) injection. Since these two mouse strains only differ in whether or not they have the ASGP receptor, it can be concluded that proper ASGP receptor function exerted a protective effect against CCl(4) toxicity. Thus, receptor-mediated endocytosis by the ASGP receptor could represent a novel molecular mechanism that is responsible for subsequent liver health or injury.

The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acid alpha 2,6GalNAc.

Endogenous ligands have not, to date, been identified for the asialoglycoprotein receptor (ASGP-R), which is abundantly expressed by parenchymal cells in the liver of mammals. On the basis of the rapid clearance of BSA bearing multiple chemically coupled sialic acid (Sia)alpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man tetrasaccharides (SiaGGnM-BSA) from the circulation, and the ability of the ASGP-R hepatic lectin-1 subunit to bind SiaGGnM-BSA, we previously proposed that glycoproteins modified with structures terminating with Siaalpha2,6GalNAc may represent previously unrecognized examples of endogenous ligands for this receptor. Here, we have taken a genetic approach using wild-type and ASGP-R-deficient mice to determine that the ASGP-R in vivo does indeed account for the rapid clearance of glycoconjugates terminating with Siaalpha2,6GalNAc. We have also determined that the ASGP-R is able to bind core-substituted oligosaccharides with the terminal sequence Siaalpha2,6Galbeta1,4GlcNAc but not those with the terminal Siaalpha2,3Galbeta1,4GlcNAc. We propose that glycoproteins bearing terminals Siaalpha2,6GalNAc and Siaalpha2,6Gal are endogenous ligands for the ASGP-R, and that the ASGP-R helps to regulate the relative concentration of serum glycoproteins bearing alpha2,6-linked Sia.