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.

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.

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.

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.

Aqueous Cichorium intybus L. seed extract may protect against acute palmitate-induced impairment in cultured human umbilical vein endothelial cells by adjusting the Akt/eNOS pathway, ROS: NO ratio and ET-1 concentration.

BACKGROUND: Endothelial dysfunction, which is a vascular response to oxidative stress and inflammation, involves a cascade of downstream events that lead to decreased synthesis of insulin-mediated vasodilator nitric oxide (NO) and increased production of vasoconstrictor protein endothelin-1 (ET-1). NO, and ET-1 production by endothelial cells is regulated by phosphatidylinositol 3-kinase (PI3K)-Akt-eNOS axis and mitogen-activated protein kinase (MAPK) axis of the insulin signaling pathway, respectively. METHODS: After treating the human umbilical vein endothelial cells (HUVECs) with either palmitate complexed with bovine serum albumin (BSA) (abbreviated as PA) or the aqueous Cichorium intybus L. (chicory) seed extract (chicory seed extract, abbreviated as CSE) alone, and simultaneously together (PA + CSE), for 3, 12, and 24 h, we evaluated the capacity of CSE to reestablish the PA-induced imbalance between PI3K/Akt/eNOS and MAPK signaling pathways. The level of oxidative stress was determined by fluorimeter. Insulin-induced levels of NO and ET-1 were measured by Griess and ELISA methods, respectively. Western blotting was used to determine the extent of Akt and eNOS phosphorylation. RESULTS: Contrary to PA that caused an increase in the reactive oxygen species (ROS) levels and attenuated NO production, CSE readjusted the NO/ROS ratio within 12 h. CSE improved the metabolic arm of the insulin signaling pathway by up-regulating the insulin-stimulated phospho-eNOS Ser1177/total eNOS and phospho-Akt Thr308/total Akt ratios and decreased ET-1 levels. CONCLUSIONS: CSE ameliorated the PA-induced endothelial dysfunction not only by its anti-ROS property but also by selectively enhancing the protective arm and diminishing the injurious arm of insulin signaling pathways.