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+Cc1(fl/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+Cc1(fl/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-kappaB and increase the transcription of matrix metalloproteases, ultimately inducing plasma IL-6 and TNFalpha 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

Tissue-selective estrogen complexes with bazedoxifene prevent metabolic dysfunction in female mice

Pairing the selective estrogen receptor modulator bazedoxifene (BZA) with estrogen as a tissue-selective estrogen complex (TSEC) is a novel menopausal therapy. We investigated estrogen, BZA and TSEC effects in preventing diabetisity in ovariectomized mice during high-fat feeding. Estrogen, BZA or TSEC prevented fat accumulation in adipose tissue, liver and skeletal muscle, and improved insulin resistance and glucose intolerance without stimulating uterine growth. Estrogen, BZA and TSEC improved energy homeostasis by increasing lipid oxidation and energy expenditure, and promoted insulin action by enhancing insulin-stimulated glucose disposal and suppressing hepatic glucose production. While estrogen improved metabolic homeostasis, at least partially, by increasing hepatic production of FGF21, BZA increased hepatic expression of Sirtuin1, PPARα and AMPK activity. The metabolic benefits of BZA were lost in estrogen receptor-α deficient mice. Thus, BZA alone or in TSEC produces metabolic signals of fasting and caloric restriction and improves energy and glucose homeostasis in female mice

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

The study was partially presented as a poster in the 53rd Annual Meeting of the European Association for the Study of Diabetes, Lisbon 2017.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.This work was supported by grants from the Ministerio de Economía, Industria y Competitividad: SAF2014-58702-C2-1-R and SAF2016-77871-C2-1-R to ICC; SAF2014-58702-C2-2-R and SAF2016-77871-C2-2-R to GP; supported by the EFSD European Research Programme on New Targets for Type 2 Diabetes supported by an educational research grant from MSD to ICC and GP; the National Institutes of Health: R01-DK054254, R01-DK083850 and RO1-HL-112248 to SMN, and R01-GM115617 to MAL; and the American Diabetes Association: Career Development Award 7-11-CD-13 to MAL.Peer reviewe

Tumor and Endothelial Cell-Derived Microvesicles Carry Distinct CEACAMs and Influence T-Cell Behavior

Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1 000 nm in diameter), which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter), derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM)-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis

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

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

Introduction of a filtration step into the MVs isolation protocol significantly improves pureness of the isolates.

<p>A) Transmission electron micrographs of MVs isolated from serum starved HT29 supernatants by centrifugation without (a) or with (b) prior 0.8 µm filtration step. Samples were fixed, embedded in mold, cut in thin sections and observed using transmission electron microscopy. B) MVs filtered and isolated from serum starved HT29 cells were analyzed with the nanoparticle tracking analysis, allowing estimating the average size distribution of MVs.</p

Pervanadate and peroxide treatment induce tyrosine phosphorylation in cells but not in corresponding MVs.

<p>Confluent HT29 cells and corresponding MVs were treated with pervanadate or H₂O₂, respectively, or left untreated. Western blot analyzes were performed using mAb 4G10 for detecting tyrosine phosphorylation in A) the CEACAM1 immunoprecipitates, and B) the whole cell and MVs lysates. In A) CEACAM1 detection and in B) beta-actin detection served as loading control. The data show one representative result of three independent repeats of the experiment.</p

Colon tumor epithelial cells release MVs in response to serum starvation.

<p>A) Transmission electron micrographs of HT29 cells grown under normal conditions (a) and serum starved (b) for 48 h. After culture, cells fixed, embedded in mold, cut in thin sections and analyzed by transmission electron microscopy. B) The amount of MVs released by HT29 cells cultured in media with or without serum was counted by flow cytometry (n=3).</p

CEACAM1-positive MVs significantly increase the anti-CD3 and anti-CD3/CD28 mAb triggered T-cell proliferation.

<p>Freshly isolated human PBMC were labeled with CFSE and cultured for 4 days in the presence of anti CD3 and anti CD3/CD28 with and without CHO- and CHO-CEACAM1 derived MVs (A). B) CFSE labeled PBMC were cultured for 4 days in the presence and absence of antiCD3 and antiCD3/CD28 with and without HT29-derived MVs. Untreated treated cells served as control. In indicated cases samples were co-cultured with antiCEACAM1 mAb18/20 (50 µg/ml) or isotype matched control IgG (50 µg/ml). Then PBMCs were analyzed utilizing the Accuri C6 flow cytometer system. The histograms depict PBMCs that have divided 1-3 times based on CFSE dilution peaks and reflex the cell proliferation rate given in %. The data shown are representative for three independent repeats of the experiment.</p