Loss of CEACAM1 in hepatocytes causes hepatic fibrosis.

BACKGROUND: The role of insulin resistance in hepatic fibrosis in Metabolic dysfunction-Associated SteatoHepatitis (MASH) remains unclear. Carcinoembryonic Antigen-related Cell Adhesion Molecule1 protein (CEACAM1) promotes insulin clearance to maintain insulin sensitivity and repress de novo lipogenesis, as bolstered by the development of insulin resistance and steatohepatitis in AlbuminCre + Cc1fl/fl mice with liver-specific mouse gene encoding CEACAM1 protein (Ceacam1) deletion. We herein investigated whether these mice also developed hepatic fibrosis and whether hepatic CEACAM1 is reduced in patients with MASH at different fibrosis stages. METHODS: AlbuminCre + Cc1fl/fl mice were fed a regular or a high-fat diet before their insulin metabolism and action were assessed during IPGTT, and their livers excised for histochemical, immunohistochemical and Western blot analysis. Sirius red staining was used to assess fibrosis, and media transfer was employed to examine whether mutant hepatocytes activated hepatic stellate cells (HSCs). Hepatic CEACAM1 protein levels in patients with varying disease stages were assessed by ELISA. RESULTS: Hepatocytic deletion of Ceacam1 caused hyperinsulinemia-driven insulin resistance emanating from reduced hepatic insulin clearance. AlbuminCre + Cc1fl/fl livers showed inflammation, fibrosis and hepatic injury, with more advanced bridging and chicken-wire hepatic fibrosis under high-fat conditions. Media transferred from hepatocytes isolated from mutant mice activated control HSCs, likely owing to their elevated endothelin1 content. Interestingly, hepatic CEACAM1 levels were lower in the livers of patients with MASH and declined gradually with advanced fibrosis stage. CONCLUSIONS: Hepatic CEACAM1 levels declined with progression of MASH in humans. The phenotype of AlbuminCre + Cc1fl/fl mice assigned a key role to CEACAM1 loss from hepatocytes in hepatic fibrosis independently of other liver cells.

Cholesterol depletion decreases adhesion of non-small cell lung cancer cells to E-selectin.

Lipid microdomains, ordered membrane phases containing cholesterol and glycosphingolipids, play an essential role in cancer cell adhesion and ultimately metastasis. Notably, elevated levels of cholesterol-rich lipid microdomains are found in cancer cells relative to their normal counterparts. Therefore, alterations of lipid microdomains through cholesterol modulation could be used as a strategy to prevent cancer metastasis. In this study, methyl-beta-cyclodextrin (MßCD), sphingomyelinase (SMase), and simvastatin (Simva) were used to investigate the effects of cholesterol on the adhesive behaviors of four non-small cell lung cancer (NSCLC) cell lines (H1299, H23, H460, and A549) and a small cell lung cancer (SCLC) cell line (SHP-77) on E-selectin, a vascular endothelial molecule that initiates circulating tumor cell recruitment at metastatic sites. Under hemodynamic flow conditions, the number of adherent NSCLC cells on E-selectin significantly decreased by MßCD and Simva treatments, whereas SMase treatment did not show a significant effect. Significant increases in rolling velocities were detected only for H1299 and H23 cells after MßCD treatment. In contrast, cholesterol depletion did not affect SCLC cell attachment and rolling velocities. Moreover, cholesterol depletion by MßCD and Simva induced CD44 shedding and resulted in an enhanced membrane fluidity in the NSCLC cells, whereas it did not affect the membrane fluidity of the SCLC cells which lacked detectable expression of CD44. Our finding suggests that cholesterol regulates the E-selectin-mediated adhesion of NSCLC cells by redistributing the CD44 glycoprotein and thus modulating the membrane fluidity.NEW & NOTEWORTHY This study investigates the effects of cholesterol on the adhesive behaviors of lung cancer cells in recruitment at metastatic sites. Using cholesterol-modulating compounds, we found that reducing cholesterol decreases the adhesion of non-small cell lung cancer (NSCLC) cells while having no significant effect on small cell lung cancer (SCLC) cells. The study suggests that cholesterol regulates NSCLC cell metastasis by redistributing the adhesion proteins on the cells and modulating cells' membrane fluidity.

Aortic Fibrosis in Insulin-Sensitive Mice with Endothelial Cell-Specific Deletion of Ceacam1 Gene.

(1) Background: Mice with global Ceacam1 deletion developed plaque-like aortic lesions even on C57BL/6J background in the presence of increased endothelial cell permeability and insulin resistance. Loss of endothelial Ceacam1 gene caused endothelial dysfunction and reduced vascular integrity without affecting systemic insulin sensitivity. Because endothelial cell injury precedes atherosclerosis, we herein investigated whether the loss of endothelial Ceacam1 initiates atheroma formation in the absence of insulin resistance. (2) Methods: Endothelial cell-specific Ceacam1 null mice on C57BL/6J.Ldlr-/- background (Ldlr-/-VECadCre+Cc1fl/fl) were fed an atherogenic diet for 3-5 months before metabolic, histopathological, and en-face analysis of aortae were compared to their control littermates. Sirius Red stain was also performed on liver sections to analyze hepatic fibrosis. (3) Results: These mice displayed insulin sensitivity without significant fat deposition on aortic walls despite hypercholesterolemia. They also displayed increased inflammation and fibrosis. Deleting Ceacam1 in endothelial cells caused hyperactivation of VEGFR2/Shc/NF-κB pathway with resultant transcriptional induction of NF-κB targets. These include IL-6 that activates STAT3 inflammatory pathways, in addition to endothelin-1 and PDGF-B profibrogenic effectors. It also induced the association between SHP2 phosphatase and VEGFR2, downregulating the Akt/eNOS pathway and reducing nitric oxide production, a characteristic feature of endothelial dysfunction. Similarly, hepatic inflammation and fibrosis developed in Ldlr-/-VECadCre+Cc1fl/fl mice without an increase in hepatic steatosis. (4) Conclusions: Deleting endothelial cell Ceacam1 caused hepatic and aortic inflammation and fibrosis with increased endothelial dysfunction and oxidative stress in the presence of hypercholesterolemia. However, this did not progress into frank atheroma formation. Because these mice remained insulin sensitive, the study provides an in vivo demonstration that insulin resistance plays a critical role in the pathogenesis of frank atherosclerosis.

Nicotinamide Mononucleotide Prevents Free Fatty Acid-Induced Reduction in Glucose Tolerance by Decreasing Insulin Clearance.

The NAD-dependent deacetylase SIRT1 improves ß cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents ß cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on ß cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced ß cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased ß cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.

Age-dependent insulin resistance in male mice with null deletion of the carcinoembryonic antigen-related cell adhesion molecule 2 gene.

AIMS/HYPOTHESIS: Cc2 -/- mice lacking the gene encoding the carcinoembryonic-antigen-related cell adhesion molecule 2 (Cc2 [also known as Ceacam2]) exhibit hyperphagia that leads to obesity and insulin resistance. This starts at 2 months of age in female mice. Male mutants maintain normal body weight and insulin sensitivity until the last age previously examined (7-8 months), owing to increased sympathetic tone to white adipose tissue and energy expenditure. The current study investigates whether insulin resistance develops in mutant male mice at a later age and whether this is accompanied by changes in insulin homeostasis. METHODS: Insulin response was assessed by insulin and glucose tolerance tests. Energy balance was analysed by indirect calorimetry. RESULTS: Male Cc2 -/- mice developed overt metabolic abnormalities at about 9 months of age. These include elevated global fat mass, hyperinsulinaemia and insulin resistance (as determined by glucose and insulin intolerance, fed hyperglycaemia and decreased insulin signalling pathways). Pair-feeding experiments showed that insulin resistance resulted from hyperphagia. Indirect calorimetry demonstrated that older mutant male mice had compromised energy expenditure. Despite increased insulin secretion caused by Cc2 deletion, chronic hyperinsulinaemia did not develop in mutant male mice until about 9 months of age, at which point insulin clearance began to decline substantially. This was probably mediated by a marked decrease in hepatic CEACAM1 expression. CONCLUSIONS/INTERPRETATION: The data demonstrate that at about 9 months of age, Cc2 -/- male mice develop a reduction in energy expenditure and energy imbalance which, combined with a progressive decrease in CEACAM1-dependent hepatic insulin clearance, causes chronic hyperinsulinaemia and sustained age-dependent insulin resistance. This represents a novel mechanistic underpinning of age-related impairment of hepatic insulin clearance.

Liver-specific reconstitution of CEACAM1 reverses the metabolic abnormalities caused by its global deletion in male mice.

AIMS/HYPOTHESIS: The carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes insulin clearance. Mice with global null mutation (Cc1 -/-) or with liver-specific inactivation (L-SACC1) of Cc1 (also known as Ceacam1) gene display hyperinsulinaemia resulting from impaired insulin clearance, insulin resistance, steatohepatitis and obesity. Because increased lipolysis contributes to the metabolic phenotype caused by transgenic inactivation of CEACAM1 in the liver, we aimed to further investigate the primary role of hepatic CEACAM1-dependent insulin clearance in insulin and lipid homeostasis. To this end, we examined whether transgenic reconstitution of CEACAM1 in the liver of global Cc1 -/- mutant mice reverses their abnormal metabolic phenotype. METHODS: Insulin response was assessed by hyperinsulinaemic-euglycaemic clamp analysis and energy balance was analysed by indirect calorimetry. Mice were overnight-fasted and refed for 7 h to assess fatty acid synthase activity in the liver and the hypothalamus in response to insulin release during refeeding. RESULTS: Liver-based rescuing of CEACAM1 restored insulin clearance, plasma insulin level, insulin sensitivity and steatohepatitis caused by global deletion of Cc1. It also reversed the gain in body weight and total fat mass observed with Cc1 deletion, in parallel to normalising energy balance. Mechanistically, reversal of hyperphagia appeared to result from reducing fatty acid synthase activity and restoring insulin signalling in the hypothalamus. CONCLUSIONS/INTERPRETATION: Despite the potential confounding effects of deleting Cc1 from extrahepatic tissues, liver-based rescuing of CEACAM1 resulted in full normalisation of the metabolic phenotype, underscoring the key role that CEACAM1-dependent hepatic insulin clearance pathways play in regulating systemic insulin sensitivity, lipid homeostasis and energy balance.

PPARα (Peroxisome Proliferator-activated Receptor α) Activation Reduces Hepatic CEACAM1 Protein Expression to Regulate Fatty Acid Oxidation during Fasting-refeeding Transition.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is expressed at high levels in the hepatocyte, consistent with its role in promoting insulin clearance in liver. CEACAM1 also mediates a negative acute effect of insulin on fatty acid synthase activity. Western blot analysis reveals lower hepatic CEACAM1 expression during fasting. Treating of rat hepatoma FAO cells with Wy14,643, an agonist of peroxisome proliferator-activated receptor α (PPARα), rapidly reduces Ceacam1 mRNA and CEACAM1 protein levels within 1 and 2 h, respectively. Luciferase reporter assay shows a decrease in the promoter activity of both rat and mouse genes by Pparα activation, and 5'-deletion and block substitution analyses reveal that the Pparα response element between nucleotides -557 and -543 is required for regulation of the mouse promoter activity. Chromatin immunoprecipitation analysis demonstrates binding of liganded Pparα toCeacam1promoter in liver lysates ofPparα(+/+), but notPparα(-/-)mice fed a Wy14,643-supplemented chow diet. Consequently, Wy14,643 feeding reduces hepatic Ceacam1 mRNA and CEACAM1 protein levels, thus decreasing insulin clearance to compensate for compromised insulin secretion and maintain glucose homeostasis and insulin sensitivity in wild-type mice. Together, the data show that the low hepatic CEACAM1 expression at fasting is mediated by Pparα-dependent mechanisms. Changes in CEACAM1 expression contribute to the coordination of fatty acid oxidation and insulin action in the fasting-refeeding transition.

Conditional deletion of CEACAM1 causes hepatic stellate cell activation.

Objectives: Hepatic CEACAM1 expression declines with advanced hepatic fibrosis stage in patients with MASH. Global and hepatocyte-specific deletions of Ceacam1 impair insulin clearance to cause hepatic insulin resistance and steatosis. They also cause hepatic inflammation and fibrosis, a condition characterized by excessive collagen production from activated hepatic stellate cells (HSCs). Given the positive effect of PPARγ on CEACAM1 transcriptoin and on HSCs quiescence, the current studies investigated whether CEACAM1 loss from HSCs causes their activation. Methods: We examined whether lentiviral shRNA-mediated CEACAM1 donwregulation (KD-LX2) activates cultured human LX2 stellate cells. We also generated LratCre+Cc1 fl/fl mutants with conditional Ceacam1 deletion in HSCs and characterized their MASH phenotype. Media transfer experiments were employed to examine whether media from mutant human and murine HSCs activate their wild-type counterparts. Results: LratCre+Cc1 fl/fl mutants displayed hepatic inflammation and fibrosis but without insulin resistance or hepatic steatosis. Their HSCs, like KD-LX2 cells, underwent myofibroblastic transformation and their media activated wild-type HDCs. This was inhibited by nicotinic acid treatment which stemmed the release of IL-6 and fatty acids, both of which activate the epidermal growth factor receptor (EGFR) tyrosine kinase. Gefitinib inhibition of EGFR and its downstream NF-κB/IL-6/STAT3 inflammatory and MAPK-proliferation pathways also blunted HSCs activation in the absence of CEACAM1. Conclusions: Loss of CEACAM1 in HSCs provoked their myofibroblastic transformation in the absence of insulin resistance and hepatic steatosis. This response is mediated by autocrine HSCs activation of the EGFR pathway that amplifies inflammation and proliferation.

Conditional deletion of CEACAM1 in hepatic stellate cells causes their activation.

OBJECTIVES: Hepatic CEACAM1 expression declines with advanced hepatic fibrosis stage in patients with metabolic dysfunction-associated steatohepatitis (MASH). Global and hepatocyte-specific deletions of Ceacam1 impair insulin clearance to cause hepatic insulin resistance and steatosis. They also cause hepatic inflammation and fibrosis, a condition characterized by excessive collagen production from activated hepatic stellate cells (HSCs). Given the positive effect of PPARγ on CEACAM1 transcription and on HSCs quiescence, the current studies investigated whether CEACAM1 loss from HSCs causes their activation. METHODS: We examined whether lentiviral shRNA-mediated CEACAM1 donwregulation (KD-LX2) activates cultured human LX2 stellate cells. We also generated LratCre + Cc1fl/fl mutants with conditional Ceacam1 deletion in HSCs and characterized their MASH phenotype. Media transfer experiments were employed to examine whether media from mutant human and murine HSCs activate their wild-type counterparts. RESULTS: LratCre + Cc1fl/fl mutants displayed hepatic inflammation and fibrosis but without insulin resistance or hepatic steatosis. Their HSCs, like KD-LX2 cells, underwent myofibroblastic transformation and their media activated wild-type HSCs. This was inhibited by nicotinic acid treatment which blunted the release of IL-6 and fatty acids, both of which activate the epidermal growth factor receptor (EGFR) tyrosine kinase. Gefitinib inhibition of EGFR and its downstream NF-κB/IL-6/STAT3 inflammatory and MAPK-proliferation pathways also blunted HSCs activation in the absence of CEACAM1. CONCLUSIONS: Loss of CEACAM1 in HSCs provoked their myofibroblastic transformation in the absence of insulin resistance and hepatic steatosis. This response is mediated by autocrine HSCs activation of the EGFR pathway that amplifies inflammation and proliferation.

Ceacam1 deletion causes vascular alterations in large vessels.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulin's stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Loss of CEACAM1 in endothelial cells causes hepatic fibrosis.

OBJECTIVES: Hepatocytic CEACAM1 plays a critical role in NASH pathogenesis, as bolstered by the development of insulin resistance, visceral obesity, steatohepatitis and fibrosis in mice with global Ceacam1 (Cc1) deletion. In contrast, VECadCre+Cc1fl/fl mice with endothelial loss of Cc1 manifested insulin sensitivity with no visceral obesity despite elevated NF-κB signaling and increased systemic inflammation. We herein investigated whether VECadCre+Cc1fl/fl male mice develop hepatic fibrosis and whether this is mediated by increased production of endothelin1 (ET1), a transcriptional NF-κB target. METHODS: VECadCre+Et1.Cc1fl/fl mice with combined endothelial loss of Cc1/Et1 genes were generated. Histological and immunohistochemical analyses were conducted on their livers and on liver tissue biopsies from adult patients undergoing bariatric surgery or from patients with NASH diagnosis receiving liver transplant. RESULTS: Hepatic fibrosis and inflammatory infiltration developed in VECadCre+Cc1fl/fl liver parenchyma. This was preceded by increased ET1 production and reversed with combined endothelial loss of Et1. Conditioned media from VECadCre+Cc1fl/fl, but not VECadCre+Et1.Cc1fl/fl primary liver endothelial cells activated wild-type hepatic stellate cells; a process inhibited by bosentan, an ETAR/ETBR dual antagonist. Consistently, immunohistochemical analysis of liver biopsies from patients with NASH showed a decline in endothelial CEACAM1 in parallel with increased plasma endothelin1 levels and progression of hepatic fibrosis stage. CONCLUSIONS: The data demonstrated that endothelial CEACAM1 plays a key role in preventing hepatic fibrogenesis by reducing autocrine endothelin1 production.

Regulation of Insulin Clearance by Non-Esterified Fatty Acids.

Insulin stores lipid in adipocytes and prevents lipolysis and the release of non-esterified fatty acids (NEFA). Excessive release of NEFA during sustained energy supply and increase in abdominal adiposity trigger systemic insulin resistance, including in the liver, a major site of insulin clearance. This causes a reduction in insulin clearance as a compensatory mechanism to insulin resistance in obesity. On the other hand, reduced insulin clearance in the liver can cause chronic hyperinsulinemia, followed by downregulation of insulin receptor and insulin resistance. Delineating the cause-effect relationship between reduced insulin clearance and insulin resistance has been complicated by the fact that insulin action and clearance are mechanistically linked to insulin binding to its receptors. This review discusses how NEFA mobilization contributes to the reciprocal relationship between insulin resistance and reduced hepatic insulin clearance, and how this may be implicated in the pathogenesis of non-alcoholic fatty liver disease.

Regulation of hepatic fibrosis by carcinoembryonic antigen-related cell adhesion molecule 1.

OBJECTIVE: NAFLD is a complex disease marked by cellular abnormalities leading to NASH. NAFLD patients manifest low hepatic levels of CEACAM1, a promoter of insulin clearance. Consistently, Cc1-/- null mice displayed spontaneous hyperinsulinemia/insulin resistance and steatohepatitis. Liver-specific reconstitution of Ceacam1 reversed these metabolic anomalies in 8-month-old Cc1-/-xliver+ mice fed a regular chow diet. The current study examined whether it would also reverse progressive hepatic fibrosis in mice fed a high-fat (HF) diet. METHODS: 3-Month-old mice were fed a high-fat diet for 3-5 months, and metabolic and histopathological analysis were conducted to evaluate their NASH phenotype. RESULTS: Reconstituting CEACAM1 to Cc1-/- livers curbed diet-induced liver dysfunction and NASH, including macrovesicular steatosis, lobular inflammation, apoptosis, oxidative stress, and chicken-wire bridging fibrosis. Persistence of hepatic fibrosis in HF-fed Cc1-/- treated with nicotinic acid demonstrated a limited role for lipolysis and adipokine release in hepatic fibrosis caused by Ceacam1 deletion. CONCLUSIONS: Restored metabolic and histopathological phenotype of HF-fed Cc1-/-xliver+xliver+ assigned a critical role for hepatic CEACAM1 in preventing NAFLD/NASH including progressive hepatic fibrosis.

Insulin Sensitivity Is Retained in Mice with Endothelial Loss of Carcinoembryonic Antigen Cell Adhesion Molecule 1.

CEACAM1 regulates endothelial barrier integrity. Because insulin signaling in extrahepatic target tissues is regulated by insulin transport through the endothelium, we aimed at investigating the metabolic role of endothelial CEACAM1. To this end, we generated endothelial cell-specific Ceacam1 null mice (VECadCre+Cc1fl/fl) and carried out their metabolic phenotyping and mechanistic analysis by comparison to littermate controls. Hyperinsulinemic-euglycemic clamp analysis showed intact insulin sensitivity in VECadCre+Cc1fl/fl mice. This was associated with the absence of visceral obesity and lipolysis and normal levels of circulating non-esterified fatty acids, leptin, and adiponectin. Whereas the loss of endothelial Ceacam1 did not affect insulin-stimulated receptor phosphorylation, it reduced IRS-1/Akt/eNOS activation to lower nitric oxide production resulting from limited SHP2 sequestration. It also reduced Shc sequestration to activate NF-κB and increase the transcription of matrix metalloproteases, ultimately inducing plasma IL-6 and TNFα levels. Loss of endothelial Ceacam1 also induced the expression of the anti-inflammatory CEACAM1-4L variant in M2 macrophages in white adipose tissue. Together, this could cause endothelial barrier dysfunction and facilitate insulin transport, sustaining normal glucose homeostasis and retaining fat accumulation in adipocytes. The data assign a significant role for endothelial cell CEACAM1 in maintaining insulin sensitivity in peripheral extrahepatic target tissues.

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

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+ Cc1flox ( 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+Cc1fl/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.

Loss of Ceacam1 promotes prostate cancer progression in Pten haploinsufficient male mice.

OBJECTIVE: PTEN haploinsufficiency plays an important role in prostate cancer development in men. However, monoallelic deletion of Pten gene failed to induce high prostate intraepithelial neoplasia (PIN) until Pten+/- mice aged or fed a high-calorie diet. Because CEACAM1, a cell adhesion molecule with a potential tumor suppression activity, is induced in Pten+/- prostates, the study aimed at examining whether the rise of CEACAM1 limited neoplastic progression in Pten+/- prostates. METHODS: Pten+/- were crossbred with Cc1-/- mice harboring a null deletion of Ceacam1 gene to produce Pten+/-/Cc1-/- double mutants. Prostates from 7-month old male mice were analyzed histologically and biochemically for PIN progression. RESULTS: Deleting Ceacam1 in Pten+/- mice caused an early development of high-grade PIN in parallel to hyperactivation of PI3 kinase/Akt and Ras/MAP kinase pathways, with an increase in cell proliferation, epithelial-to-mesenchymal transition, angiogenesis and inflammation relative to Pten+/- and Cc1-/- individual mutants. It also caused a remarkable increase in lipogenesis in prostate despite maintaining insulin sensitivity. Concomitant Ceacam1 deletion with Pten+/- activated the IL-6/STAT3 signaling pathways to suppress Irf-8 transcription that in turn, led to a decrease in the expression level of promyelocytic leukemia gene, a well characterized tumor suppressor in prostate. CONCLUSIONS: Ceacam1 deletion accelerated high-grade prostate intraepithelial neoplasia in Pten haploinsufficient mice while preserving insulin sensitivity. This demonstrated that the combined loss of Ceacam1 and Pten advanced prostate cancer by increasing lipogenesis and modifying the STAT3-dependent inflammatory microenvironment of prostate.

Hyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis.

BACKGROUND: CEACAM1 regulates insulin sensitivity by promoting insulin clearance. Accordingly, global C57BL/6J.Cc1-/- null mice display hyperinsulinemia due to impaired insulin clearance at 2 months of age, followed by insulin resistance, steatohepatitis, visceral obesity and leptin resistance at 6 months. The study aimed at investigating the primary role of hepatic CEACAM1 in insulin and lipid homeostasis independently of its metabolic effect in extra-hepatic tissues. METHODS: Liver-specific C57BL/6J.AlbCre+Cc1fl/fl mice were generated and their metabolic phenotype was characterized by comparison to that of their littermate controls at 2-9 months of age, using hyperinsulinemic-euglycemic clamp analysis and indirect calorimetry. The effect of hyperphagia on insulin resistance was assessed by pair-feeding experiments. RESULTS: Liver-specific AlbCre+Cc1fl/fl mutants exhibited impaired insulin clearance and hyperinsulinemia at 2 months, followed by hepatic insulin resistance (assessed by hyperinsulinemic-euglycemic clamp analysis) and steatohepatitis at ~ 7 months of age, at which point visceral obesity and hyperphagia developed, in parallel to hyperleptinemia and blunted hypothalamic STAT3 phosphorylation in response to an intraperitoneal injection of leptin. Hyperinsulinemia caused hypothalamic insulin resistance, followed by increased fatty acid synthase activity, which together with defective hypothalamic leptin signaling contributed to hyperphagia and reduced physical activity. Pair-feeding experiment showed that hyperphagia caused systemic insulin resistance, including blunted insulin signaling in white adipose tissue and lipolysis, at 8-9 months of age. CONCLUSION: AlbCre+Cc1fl/fl mutants provide an in vivo demonstration of the key role of impaired hepatic insulin clearance and hyperinsulinemia in the pathogenesis of secondary hepatic insulin resistance independently of lipolysis. They also reveal an important role for the liver-hypothalamic axis in the regulation of energy balance and subsequently, systemic insulin sensitivity.

Liver-specific ablation of insulin-degrading enzyme causes hepatic insulin resistance and glucose intolerance, without affecting insulin clearance in mice.

The role of insulin-degrading enzyme (IDE), a metalloprotease with high affinity for insulin, in insulin clearance remains poorly understood. OBJECTIVE: This study aimed to clarify whether IDE is a major mediator of insulin clearance, and to define its role in the etiology of hepatic insulin resistance. METHODS: We generated mice with liver-specific deletion of Ide (L-IDE-KO) and assessed insulin clearance and action. RESULTS: L-IDE-KO mice exhibited higher (~20%) fasting and non-fasting plasma glucose levels, glucose intolerance and insulin resistance. This phenotype was associated with ~30% lower plasma membrane insulin receptor levels in liver, as well as ~55% reduction in insulin-stimulated phosphorylation of the insulin receptor, and its downstream signaling molecules, AKT1 and AKT2 (reduced by ~40%). In addition, FoxO1 was aberrantly distributed in cellular nuclei, in parallel with up-regulation of the gluconeogenic genes Pck1 and G6pc. Surprisingly, L-IDE-KO mice showed similar plasma insulin levels and hepatic insulin clearance as control mice, despite reduced phosphorylation of the carcinoembryonic antigen-related cell adhesion molecule 1, which upon its insulin-stimulated phosphorylation, promotes receptor-mediated insulin uptake to be degraded. CONCLUSION: IDE is not a rate-limiting regulator of plasma insulin levels in vivo.

Liver-specific rescuing of CEACAM1 reverses endothelial and cardiovascular abnormalities in male mice with null deletion of Ceacam1 gene.

OBJECTIVE: Mice with global null mutation of Ceacam1 (Cc1-/-), display impairment of insulin clearance that causes hyperinsulinemia followed by insulin resistance, elevated hepatic de novo lipogenesis, and visceral obesity. In addition, they manifest abnormal vascular permeability and elevated blood pressure. Liver-specific rescuing of Ceacam1 reversed all of the metabolic abnormalities in Cc1-/-liver+ mice. The current study examined whether Cc1-/- male mice develop endothelial and cardiac dysfunction and whether this relates to the metabolic abnormalities caused by defective insulin extraction. METHODS AND RESULTS: Myography studies showed reduction of agonist-stimulated nitric oxide production in resistance arterioles in Cc1-/-, but not Cc1-/-liver+ mice. Liver-based rescuing of CEACAM1 also attenuated the abnormal endothelial adhesiveness to circulating leukocytes in parallel to reducing plasma endothelin-1 and recovering plasma nitric oxide levels. Echocardiography studies revealed increased septal wall thickness, cardiac hypertrophy and reduced cardiac performance in Cc1-/-, but not Cc1-/-xliver+ mice. Insulin signaling experiments indicated compromised IRS1/Akt/eNOS pathway leading to lower nitric oxide level, and activated Shc/MAPK pathway leading to more endothelin-1 production in the aortae and hearts of Cc1-/-, but not Cc1-/-xliver+ mice. The increase in the ratio of endothelin-1 receptor A/B indicated an imbalance in the vasomotor activity of Cc1-/- mice, which was normalized in Cc1-/-xliver+ mice. CONCLUSIONS: The data underscore a critical role for impaired CEACAM1-dependent hepatic insulin clearance pathways and resulting hyperinsulinemia and lipid accumulation in aortae and heart in regulating the cardiovascular function.

Exenatide induces carcinoembryonic antigen-related cell adhesion molecule 1 expression to prevent hepatic steatosis.

Exenatide, a glucagon-like peptide-1 receptor agonist, induces insulin secretion. Its role in insulin clearance has not been adequately examined. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance to maintain insulin sensitivity. Feeding C57BL/6J mice a high-fat diet down-regulates hepatic Ceacam1 transcription to cause hyperinsulinemia, insulin resistance, and hepatic steatosis, as in Ceacam1 null mice (Cc1-/- ). Thus, we tested whether exenatide regulates Ceacam1 expression in high-fat diet-fed mice and whether this contributes to its insulin sensitizing effect. Exenatide (100 nM) induced the transcriptional activity of wild-type Ceacam1 promoter but not the constructs harboring block mutations of peroxisome proliferator-activated receptor response element and retinoid X receptor alpha, individually or collectively, in HepG2 human hepatoma cells. Chromatin immunoprecipitation analysis demonstrated binding of peroxisome proliferator-activated receptor gamma to Ceacam1 promoter in response to rosiglitazone and exenatide. Consistently, exenatide induced Ceacam1 messenger RNA expression within 12 hours in the absence but not in the presence of the glucagon-like peptide-1 receptor antagonist exendin 9-39. Exenatide (20 ng/g body weight once daily intraperitoneal injection in the last 30 days of feeding) restored hepatic Ceacam1 expression and insulin clearance to curb diet-induced metabolic abnormalities and steatohepatitis in wild-type but not Cc1-/- mice fed a high-fat diet for 2 months. Conclusion: Exenatide promotes insulin clearance in parallel with insulin secretion to prevent chronic hyperinsulinemia and the resulting hepatic steatosis, and this contributes to its insulin sensitizing effect. Our data further highlight the relevance of physiologic insulin metabolism in maintaining insulin sensitivity and normal lipid metabolism. (Hepatology Communications 2018;2:35-47).