Loss of Hepatic Carcinoembryonic Antigen‐Related Cell Adhesion Molecule 1 Links Nonalcoholic Steatohepatitis to Atherosclerosis

Abstract

Patients with nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) commonly develop atherosclerosis through a mechanism that is not well delineated. These diseases are associated with steatosis, inflammation, oxidative stress, and fibrosis. The role of insulin resistance in their pathogenesis remains controversial. Albumin ( Alb ) Cre + Cc1 flox ( fl ) /fl mice with the liver‐specific null deletion of the carcinoembryonic antigen‐related cell adhesion molecule 1 ( Ceacam1 ; alias Cc1 ) gene display hyperinsulinemia resulting from impaired insulin clearance followed by hepatic insulin resistance, elevated de novo lipogenesis, and ultimately visceral obesity and systemic insulin resistance. We therefore tested whether this mutation causes NAFLD/NASH and atherosclerosis. To this end, mice were propagated on a low‐density lipoprotein receptor ( Ldlr ) −/− background and at 4 months of age were fed a high‐cholesterol diet for 2 months. We then assessed the biochemical and histopathologic changes in liver and aortae. Ldlr −/− AlbCre + Cc1 fl/fl mice developed chronic hyperinsulinemia with proatherogenic hypercholesterolemia, a robust proinflammatory state associated with visceral obesity, elevated oxidative stress (reduced NO production), and an increase in plasma and tissue endothelin‐1 levels. In parallel, they developed NASH (steatohepatitis, apoptosis, and fibrosis) and atherosclerotic plaque lesions. Mechanistically, hyperinsulinemia caused down‐regulation of the insulin receptor followed by inactivation of the insulin receptor substrate 1–protein kinase B–endothelial NO synthase pathway in aortae, lowering the NO level. This also limited CEACAM1 phosphorylation and its sequestration of Shc‐transforming protein (Shc), activating the Shc–mitogen‐activated protein kinase–nuclear factor kappa B pathway and stimulating endothelin‐1 production. Thus, in the presence of proatherogenic dyslipidemia, hyperinsulinemia and hepatic insulin resistance driven by liver‐specific deletion of Ceacam1 caused metabolic and vascular alterations reminiscent of NASH and atherosclerosis. Conclusion: Altered CEACAM1‐dependent hepatic insulin clearance pathways constitute a molecular link between NASH and atherosclerosis.

Conditional deletion of CEACAM1 causes hepatic stellate cell activation

“…Fibrosis was assessed by staining deparaffinized and rehydrated slides with 0.1% Sirius Red stain (Direct Red 80, Sigma-Aldrich, St Louis, MO) in saturated aqueous solution of picric acid (Sigma-Aldrich), washed in 0.5% acetic acid (Sigma-Aldrich), hydrated in different alcohol percentages, and cleared in Xylene (Sigma-Aldrich). Images were taken using Nikon Eclipse 90i Microscope (Nikon, Melville, NY) as described [11]. 10 randomly selected high power fields (20x) per sample were imaged and quantified with ImageJ (v1.53t) to determine the percentage of Sirius red (SR) stain area within the whole area of imaged hepatic tissue.…”

“…Slides were then incubated with appropriate species-specific biotinylated ImmPRESS HRP horse anti-mouse or anti-rabbit secondary antibody (Vector Labs, Burlingame, CA) for 30min before being treated with 3,3’-Diaminobenzidine (DAB, Vectastain kit-Vector labs, Newark, CA) and counterstain with Mayer’s hematoxylin. Images were taken using Nikon Eclipse 90i Microscope (Nikon, Melville, NY), as previously described [11]. Sections were evaluated blindly, and positively stained cells were counted in 5 fields/mouse at 40X magnification.…”

“…Primary hepatocytes, bone marrow-derived macrophages, and endothelial cells from livers and hearts were isolated from ketamine/xylazine-anesthetized 2-to-3–month-old male mice, as previously described [11]. Briefly, hepatocytes [12] were maintained in DMEM (Gibco Lab, Gaithersburg, MD)-10% FBS, 1% glutamine, and 1% penicillin/streptomycin (Gibco).…”

“…Briefly, hepatocytes [12] were maintained in DMEM (Gibco Lab, Gaithersburg, MD)-10% FBS, 1% glutamine, and 1% penicillin/streptomycin (Gibco). Bone marrow-derived macrophages [11] were obtained by flashing femurs and tibias with sterile Roswell Park Memorial Institute (RPMI)-1640 medium from Gibco supplemented with 10% horse serum, 1% L-glutamine, 1% Penicilin/Streptomycin and 10ng/ml Recombinant Mouse factor macrophage colony-stimulating factor [M-CSF] R&D Systems, Minneapolis, MN). Primary endothelial cells [13; 14] isolated from livers and hearts were maintained in DMEM-F12 (Gibco) supplemented with 20% FBS, 1% glutamine, and 1% penicillin/streptomycin (Gibco), 100 ug/ml Heparin (Sigma-Aldrich) and 100ug/ml Endothelial Cell Growth supplement [ECGS) (EMD Millipore Corporation Burlington MA).…”