Insulin Clearance in Health and Disease.

Insulin action is impaired in type 2 diabetes. The functions of the hormone are an integrated product of insulin secretion from pancreatic ß-cells and insulin clearance by receptor-mediated endocytosis and degradation, mostly in liver (hepatocytes) and, to a lower extent, in extrahepatic peripheral tissues. Substantial evidence indicates that genetic or acquired abnormalities of insulin secretion or action predispose to type 2 diabetes. In recent years, along with the discovery of the molecular foundation of receptor-mediated insulin clearance, such as through the membrane glycoprotein CEACAM1, a consensus has begun to emerge that reduction of insulin clearance contributes to the disease process. In this review, we consider the evidence suggesting a pathogenic role for reduced insulin clearance in insulin resistance, obesity, hepatic steatosis, and type 2 diabetes.

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.

Role of Insulin Clearance in Insulin Action and Metabolic Diseases.

The year 2021 marked the centenary of the discovery of insulin [...].

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.

Hepatic Insulin Clearance: Mechanism and Physiology.

Upon its secretion from pancreatic ß-cells, insulin reaches the liver through the portal circulation to exert its action and eventually undergo clearance in the hepatocytes. In addition to insulin secretion, hepatic insulin clearance regulates the homeostatic level of insulin that is required to reach peripheral insulin target tissues to elicit proper insulin action. Receptor-mediated insulin uptake followed by its degradation constitutes the basic mechanism of insulin clearance. Upon its phosphorylation by the insulin receptor tyrosine kinase, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) takes part in the insulin-insulin receptor complex to increase the rate of its endocytosis and targeting to the degradation pathways. This review summarizes how this process is regulated and how it is associated with insulin-degrading enzyme in the liver. It also discusses the physiological implications of impaired hepatic insulin clearance: Whereas reduced insulin clearance cooperates with increased insulin secretion to compensate for insulin resistance, it can also cause hepatic insulin resistance. Because chronic hyperinsulinemia stimulates hepatic de novo lipogenesis, impaired insulin clearance also causes hepatic steatosis. Thus impaired insulin clearance can underlie the link between hepatic insulin resistance and hepatic steatosis. Delineating these regulatory pathways should lead to building more effective therapeutic strategies against metabolic syndrome.

Inhibition of protein arginine methyltransferase 5 enhances hepatic mitochondrial biogenesis.

Protein arginine methyltransferase 5 (PRMT5) regulates gene expression either transcriptionally by symmetric dimethylation of arginine residues on histones H4R3, H3R8, and H2AR3 or at the posttranslational level by methylation of nonhistone target proteins. Although emerging evidence suggests that PRMT5 functions as an oncogene, its role in metabolic diseases is not well-defined. We investigated the role of PRMT5 in promoting high-fat-induced hepatic steatosis. A high-fat diet up-regulated PRMT5 levels in the liver but not in other metabolically relevant tissues such as skeletal muscle or white and brown adipose tissue. This was associated with repression of master transcription regulators involved in mitochondrial biogenesis. In contrast, lentiviral short hairpin RNA-mediated reduction of PRMT5 significantly decreased phosphatidylinositol 3-kinase/AKT signaling in mouse AML12 liver cells. PRMT5 knockdown or knockout decreased basal AKT phosphorylation but boosted the expression of peroxisome proliferator-activated receptor α (PPARα) and PGC-1α with a concomitant increase in mitochondrial biogenesis. Moreover, by overexpressing an exogenous WT or enzyme-dead mutant PRMT5 or by inhibiting PRMT5 enzymatic activity with a small-molecule inhibitor, we demonstrated that the enzymatic activity of PRMT5 is required for regulation of PPARα and PGC-1α expression and mitochondrial biogenesis. Our results suggest that targeting PRMT5 may have therapeutic potential for the treatment of fatty liver.

CEACAM1 in Liver Injury, Metabolic and Immune Regulation.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a transmembrane glycoprotein that is expressed on epithelial, endothelial and immune cells. CEACAM1 is a differentiation antigen involved in the maintenance of epithelial polarity that is induced during hepatocyte differentiation and liver regeneration. CEACAM1 regulates insulin sensitivity by promoting hepatic insulin clearance, and controls liver tolerance and mucosal immunity. Obese insulin-resistant humans with non-alcoholic fatty liver disease manifest loss of hepatic CEACAM1. In mice, deletion or functional inactivation of CEACAM1 impairs insulin clearance and compromises metabolic homeostasis which initiates the development of obesity and hepatic steatosis and fibrosis with other features of non-alcoholic steatohepatitis, and adipogenesis in white adipose depot. This is followed by inflammation and endothelial and cardiovascular dysfunctions. In obstructive and inflammatory liver diseases, soluble CEACAM1 is shed into human bile where it can serve as an indicator of liver disease. On immune cells, CEACAM1 acts as an immune checkpoint regulator, and deletion of Ceacam1 gene in mice causes exacerbation of inflammation and hyperactivation of myeloid cells and lymphocytes. Hence, hepatic CEACAM1 resides at the central hub of immune and metabolic homeostasis in both humans and mice. This review focuses on the regulatory role of CEACAM1 in liver and biliary tract architecture in health and disease, and on its metabolic role and function as an immune checkpoint regulator of hepatic inflammation.

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.

Fenofibrate Decreases Insulin Clearance and Insulin Secretion to Maintain Insulin Sensitivity.

High fat diet reduces the expression of CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a transmembrane glycoprotein that promotes insulin clearance and down-regulates fatty acid synthase activity in the liver upon its phosphorylation by the insulin receptor. Because peroxisome proliferator-activated receptor α (PPARα) transcriptionally suppresses CEACAM1 expression, we herein examined whether high fat down-regulates CEACAM1 expression in a PPARα-dependent mechanism. By activating PPARα, the lipid-lowering drug fenofibrate reverses dyslipidemia and improves insulin sensitivity in type 2 diabetes in part by promoting fatty acid oxidation. Despite reducing glucose-stimulated insulin secretion, fenofibrate treatment does not result in insulin insufficiency. To examine whether this is mediated by a parallel decrease in CEACAM1-dependent hepatic insulin clearance pathways, we fed wild-type and Pparα-/- null mice a high fat diet supplemented with either fenofibrate or Wy14643, a selective PPARα agonist, and examined their effect on insulin metabolism and action. We demonstrated that the decrease in insulin secretion by fenofibrate and Wy14643 is accompanied by reduction in insulin clearance in wild-type but not Pparα-/- mice, thereby maintaining normoinsulinemia and insulin sensitivity despite continuous high fat intake. Intact insulin secretion in L-CC1 mice with protected hepatic insulin clearance and CEACAM1 levels provides in vivo evidence that insulin secretion responds to changes in insulin clearance to maintain physiologic insulin and glucose homeostasis. These results also emphasize the relevant role of hepatic insulin extraction in regulating insulin sensitivity.

Leptin Resistance Contributes to Obesity in Mice with Null Mutation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 1.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance. Consistently, mice with null mutation of Ceacam1 (Cc1(-/-)) exhibit impaired insulin clearance with increased lipid production in liver and redistribution to white adipose tissue, leading to visceral obesity at 2 months of age. When the mutation is propagated on the C57/BL6J genetic background, total fat mass rises significantly with age, and glucose intolerance and systemic insulin resistance develop at 6 months of age. This study was carried out to determine the mechanisms underlying the marked increase in total fat mass in 6-month-old mutants. Indirect calorimetry analysis showed that Cc1(-/-) mice develop hyperphagia and a significant reduction in physical activity, in particular in the early hours of the dark cycle, during which energy expenditure is only slightly lower than in wild-type mice. They also exhibit increased triglyceride accumulation in skeletal muscle, due in part to incomplete fatty acid ß-oxidation. Mechanistically, hypothalamic leptin signaling is reduced, as demonstrated by blunted STAT3 phosphorylation in coronal sections in response to an intracerebral ventricular injection of leptin. Hypothalamic fatty-acid synthase activity is also elevated in the mutants. Together, the data show that the increase in total fat mass in Cc1(-/-) mice is mainly attributed to hyperphagia and reduced spontaneous physical activity. Although the contribution of the loss of CEACAM1 from anorexigenic proopiomelanocortin neurons in the arcuate nucleus is unclear, leptin resistance and elevated hypothalamic fatty-acid synthase activity could underlie altered energy balance in these mice.

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.

Increased Glucose-induced Secretion of Glucagon-like Peptide-1 in Mice Lacking the Carcinoembryonic Antigen-related Cell Adhesion Molecule 2 (CEACAM2).

Carcinoembryonic antigen-related cell adhesion molecule 2 (CEACAM2) regulates food intake as demonstrated by hyperphagia in mice with the Ceacam2 null mutation (Cc2(-/-)). This study investigated whether CEACAM2 also regulates insulin secretion. Ceacam2 deletion caused an increase in ß-cell secretory function, as assessed by hyperglycemic clamp analysis, without affecting insulin response. Although CEACAM2 is expressed in pancreatic islets predominantly in non-ß-cells, basal plasma levels of insulin, glucagon and somatostatin, islet areas, and glucose-induced insulin secretion in pooled Cc2(-/-) islets were all normal. Consistent with immunofluorescence analysis showing CEACAM2 expression in distal intestinal villi, Cc2(-/-) mice exhibited a higher release of oral glucose-mediated GLP-1, an incretin that potentiates insulin secretion in response to glucose. Compared with wild type, Cc2(-/-) mice also showed a higher insulin excursion during the oral glucose tolerance test. Pretreating with exendin(9-39), a GLP-1 receptor antagonist, suppressed the effect of Ceacam2 deletion on glucose-induced insulin secretion. Moreover, GLP-1 release into the medium of GLUTag enteroendocrine cells was increased with siRNA-mediated Ceacam2 down-regulation in parallel to an increase in Ca(2+) entry through L-type voltage-dependent Ca(2+) channels. Thus, CEACAM2 regulates insulin secretion, at least in part, by a GLP-1-mediated mechanism, independent of confounding metabolic factors.

Role for hepatic CEACAM1 in regulating fatty acid metabolism along the adipocyte-hepatocyte axis.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates insulin sensitivity by promoting hepatic insulin clearance and mediating suppression of fatty acid synthase activity. Feeding C57BL/6J male mice with a high-fat (HF) diet for 3-4 weeks triggered a >60% decrease in hepatic CEACAM1 levels to subsequently impair insulin clearance and cause systemic insulin resistance and hepatic steatosis. This study aimed at investigating whether lipolysis drives reduction in hepatic CEACAM1 and whether this constitutes a key mechanism leading to diet-induced metabolic abnormalities. Blocking lipolysis with a daily intraperitoneal injection of nicotinic acid in the last two days of a 30-day HF feeding regimen demonstrated that white adipose tissue (WAT)-derived fatty acids repressed hepatic CEACAM1-dependent regulation of insulin and lipid metabolism in 3-month-old male C57BL/6J mice. Adenoviral-mediated CEACAM1 redelivery countered the adverse metabolic effect of the HF diet on insulin resistance, hepatic steatosis, visceral obesity, and energy expenditure. It also reversed the effect of HF diet on inflammation and fibrosis in WAT and liver. This assigns a causative role for lipolysis-driven decrease in hepatic CEACAM1 level and its regulation of insulin and lipid metabolism in sustaining systemic insulin resistance, hepatic steatosis, and other abnormalities associated with excessive energy supply.

CEACAM2 negatively regulates hemi (ITAM-bearing) GPVI and CLEC-2 pathways and thrombus growth in vitro and in vivo.

Carcinoembryonic antigen-related cell adhesion molecule-2 (CEACAM2) is a cell-surface glycoprotein expressed on blood, epithelial, and vascular cells. CEACAM2 possesses adhesive and signaling properties mediated by immunoreceptor tyrosine-based inhibitory motifs. In this study, we demonstrate that CEACAM2 is expressed on the surface and in intracellular pools of platelets. Functional studies of platelets from Ceacam2(-/-)-deficient mice (Cc2(-/-)) revealed that CEACAM2 serves to negatively regulate collagen glycoprotein VI (platelet) (GPVI)-FcRγ-chain and the C-type lectinlike receptor 2 (CLEC-2) signaling. Cc2(-/-) platelets displayed enhanced GPVI and CLEC-2-selective ligands, collagen-related peptide (CRP), collagen, and rhodocytin (Rhod)-mediated platelet aggregation. They also exhibited increased adhesion on type I collagen, and hyperresponsive CRP and CLEC-2-induced α and dense granule release compared with wild-type platelets. Furthermore, using intravital microscopy to ferric chloride (FeCl3)-injured mesenteric arterioles and laser-induced injury of cremaster muscle arterioles, we herein show that thrombi formed in Cc2(-/-) mice were larger and more stable than wild-type controls in vivo. Thus, CEACAM2 is a novel platelet immunoreceptor that acts as a negative regulator of platelet GPVI-collagen interactions and of ITAM receptor CLEC-2 pathways.

CEACAM2 positively regulates integrin αIIbß3-mediated platelet functions.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is an Ig-ITIM superfamily member that regulates integrin αIIbß3 function. We hypothesized that its twin protein, CEACAM2, exerts a similar physiologic role in murine platelets. CEACAM2-deficient mice (Cc2-/-) displayed prolonged tail bleeding times and increased volume of blood loss. Cc2-/- platelets have moderate integrin αIIbß3-mediated functional defects with impaired kinetics of platelet spreading on fibrinogen and type I collagen and delayed kinetics in the retraction of fibrin clots in vitro. This functional integrin αIIbß3 defect could not be attributed to altered integrin αIIbß3 expression. Cc2-/- platelets displayed normal 'inside-out' signaling properties as demonstrated by normal agonist-induced binding of soluble fluorescein isothiocyanate (FITC)-fibrinogen and JON/A antibody binding. This data provides direct evidence that disruption of CEACAM2 induces a moderate integrin αIIbß3-mediated platelet function defect, and that CEACAM2 is essential to maintain a normal integrin αIIbß3-mediated platelet function.

Differential effects of prenatal stress on metabolic programming in diet-induced obese and dietary-resistant rats.

Stress during pregnancy is a known contributing factor for the development of obesity in the offspring. Since maternal obesity is on the rise, we wanted to identify the effects of prenatal stress in the offspring of diet-induced obese (DIO) rats and compare them with the offspring of dietary-resistant (DR) rats. We hypothesized that prenatal stress would make both DIO and DR offspring susceptible to obesity, but the effect would be more pronounced in DIO rats. Pregnant DIO and DR rats were divided into two groups: nonstressed controls (control) and prenatal stress (subjected to restraint stress, three times/day from days 14 to 21 of gestation). After recording birth weight and weaning weight, male offspring were weaned onto a chow diet for 9 wk and shifted to a high-fat (HF) diet for 1 wk. At the end of the 10th wk the animals were euthanized, and visceral adipose mass, blood glucose, serum insulin, and C-peptide levels were measured. Prenatal stress resulted in hyperinsulinemia and higher C-peptide levels without altering caloric intake, body weight gain, or fat mass in the DIO offspring after 1 wk of HF intake, but not in DR offspring. To determine the mechanism underlying the hyperinsulinemia, we measured the levels of CEACAM1 that are responsible for insulin clearance. CEACAM1 levels in the liver were reduced in prenatally stressed DIO offspring after the HF challenge, suggesting that preexisting genetic predisposition in combination with prenatal stress increases the risk for obesity in adulthood, especially when offspring are fed a HF diet.

High-fat diet amplifies renal renin angiotensin system expression, blood pressure elevation, and renal dysfunction caused by Ceacam1 null deletion.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis.