A marker of endocrine receptor-positive cells, CEACAM6, is shared by two major classes of breast cancer: luminal and HER2-enriched.

Elucidating the phenotypic evolution of breast cancer through distinct subtypes relies heavily on defining a lineage blueprint of the normal human breast. Here, we show that in normal breast, within the luminal epithelial lineage, a subset of cells characterized by strong staining for endocrine receptors are also characterized by expression of the surface marker CEACAM6. Topographically, this pattern of staining predominates in terminal ductal lobular units, rather than in interlobular ducts. In culture, CEACAM6-expressing cells remain essentially postmitotic under conditions in which the other cells of luminal epithelial lineage are highly proliferative. We examined the pattern of expression among three major breast cancer subtypes: luminal, HER2-enriched, and basal-like. In 104 biopsies, the luminal and HER2-enriched subtypes showed a high proportion of CEACAM6(+) tumors (78% and 83%, respectively); the basal-like subtype showed a low proportion (28%). Further accentuation of this pattern was observed in 13 established breast cancer cell lines. When differentiation was induced by all-trans retinoic acid, CEACAM6 expression strongly correlated with luminal-like differentiation. Furthermore, CEACAM6(+) cancer cells were less proliferative than CEACAM6(-) cells in tumorsphere assays and were less tumorigenic in nude mice. Based on these observations, we propose that luminal and HER2-enriched breast cancers are more closely related than previously thought and may share a common cell of origin.

B7-H4-Ig treatment of normal mice changes lymphocyte homeostasis and increases the potential of regulatory T cells.

Enteroantigens (eAgs) drive tolerogenic and inflammatory immune responses in the gut and are of importance for sustained immune homeostasis in colonic mucosa. Decline of regulatory activity in the gut mucosa might result in chronic colitis. B7-H4 is a co-inhibitory receptor expressed by professional antigen-presenting cells. By delivering signal 2 during T cell activation, it inhibits T cell proliferation and inflammation. In this study, we have used a newly developed B7-H4-Ig fusion protein and evaluated its effect on eAg-activated effector and regulatory T cells (Treg) in vitro and in vivo. T cells were recovered from the mesenteric lymph nodes (MLNs) of untreated or B7-H4-Ig-treated BALB/c mice. Treatment of cells in vitro did neither affect the proliferation of effector T cells nor the function of Tregs. In vivo, B7-H4 treatment increased the total number of MLN-derived CD4⁺ and CD8⁺ T cell subsets as well as the functional activity of MLN-derived Tregs, whereas the proliferative activity of eAg or alloantigen specific effector T cells was not influenced, although treatment resulted in less secretion of inflammatory cytokines and chemokines from these cells. B7-H4-Ig treatment of severe combined immune-deficient (SCID) mice undergoing T cell transfer colitis did not influence the course of disease probably reflecting the lack of Tregs in this model of chronic colitis. In conclusion, we show that treatment with B7-H4-Ig in vivo changes lymphocyte homeostasis and increases the regulatory potential in normal mice, but does not affect the course of disease development in SCID mice undergoing T cell transfer colitis.

Glucagon-Like Peptide 1 and Atrial Natriuretic Peptide in a Female Mouse Model of Obstructive Pulmonary Disease.

Glucagon-like peptide-1 (GLP-1) is protective in lung disease models but the underlying mechanisms remain elusive. Because the hormone atrial natriuretic peptide (ANP) also has beneficial effects in lung disease, we hypothesized that GLP-1 effects may be mediated by ANP expression. To study this putative link, we used a mouse model of chronic obstructive pulmonary disease (COPD) and assessed lung function by unrestrained whole-body plethysmography. In 1 study, we investigated the role of endogenous GLP-1 by genetic GLP-1 receptor (GLP-1R) knockout (KO) and pharmaceutical blockade of the GLP-1R with the antagonist exendin-9 to -39 (EX-9). In another study the effects of exogenous GLP-1 were assessed. Lastly, we investigated the bronchodilatory properties of ANP and a GLP-1R agonist on isolated bronchial sections from healthy and COPD mice. Lung function did not differ between mice receiving phosphate-buffered saline (PBS) and EX-9 or between GLP-1R KO mice and their wild-type littermates. The COPD mice receiving GLP-1R agonist improved pulmonary function (P < .01) with less inflammation, but no less emphysema compared to PBS-treated mice. Compared with the PBS-treated mice, treatment with GLP-1 agonist increased ANP (nppa) gene expression by 10-fold (P < .01) and decreased endothelin-1 (P < .01), a peptide associated with bronchoconstriction. ANP had moderate bronchodilatory effects in isolated bronchial sections and GLP-1R agonist also showed bronchodilatory properties but less than ANP. Responses to both peptides were significantly increased in COPD mice (P < .05, P < .01). Taken together, our study suggests a link between GLP-1 and ANP in COPD.

Endogenous glucagon-like peptide- 1 and 2 are essential for regeneration after acute intestinal injury in mice.

OBJECTIVE: Mucositis is a side effect of chemotherapy seen in the digestive tract, with symptoms including pain, diarrhoea, inflammation and ulcerations. Our aim was to investigate whether endogenous glucagon-like peptide -1 and -2 (GLP-1 and GLP-2) are implicated in intestinal healing after chemotherapy-induced mucositis. DESIGN: We used a transgenic mouse model Tg(GCG.DTR)(Tg) expressing the human diphtheria toxin receptor in the proglucagon-producing cells. Injections with diphtheria toxin ablated the GLP-1 and GLP-2 producing L-cells in Tg mice with no effect in wild-type (WT) mice. Mice were injected with 5-fluorouracil or saline and received vehicle, exendin-4, teduglutide (gly2-GLP-2), or exendin-4/teduglutide in combination. The endpoints were body weight change, small intestinal weight, morphology, histological scoring of mucositis and myeloperoxidase levels. RESULTS: Ablation of L-cells led to impaired GLP-2 secretion; increased loss of body weight; lower small intestinal weight; lower crypt depth, villus height and mucosal area; and increased the mucositis severity score in mice given 5-fluorouracil. WT mice showed compensatory hyperproliferation as a sign of regeneration in the recovery phase. Co-treatment with exendin-4 and teduglutide rescued the body weight of the Tg mice and led to a hyperproliferation in the small intestine, whereas single treatment was less effective. CONCLUSION: The ablation of L-cells leads to severe mucositis and insufficient intestinal healing, shown by severe body weight loss and lack of compensatory hyperproliferation in the recovery phase. Co-treatment with exendin-4 and teduglutide could prevent this. Because both peptides were needed, we can conclude that both GLP-1 and GLP-2 are essential for intestinal healing in mice.

Acute administration of interleukin-6 does not increase secretion of glucagon-like peptide-1 in mice.

Interleukin 6 (IL-6) is a cytokine secreted from skeletal muscle in response to exercise which, based on animal and cell studies, has been suggested to contribute to glucose metabolism by increasing secretion of the incretin hormone glucagon-like peptide-1 (GLP-1) and affecting secretion of insulin and glucagon from the pancreatic islets. We investigated the effect of IL-6 on GLP-1 secretion in GLP-1 producing cells (GLUTag) and using the perfused mouse small intestine (harboring GLP-1 producing cells). Furthermore, the direct effect of IL-6 on insulin and glucagon secretion was studied using isolated perfused mouse pancreas. Incubating GLUTag cells with 1000 ng/mL of IL-6 for 2 h did not significantly increase secretion of GLP-1 whereas 10 mmol/L glucose (positive control) did. Similarly, IL-6 (100 ng/mL) had no effect on GLP-1 secretion from perfused mouse small intestine whereas bombesin (positive control) increased secretion. Finally, administering IL-6 (100 ng/mL) to perfused mouse pancreases did not significantly increase insulin or glucagon secretion regardless of perfusate glucose levels (3.5 vs. 12 mmol/L glucose). Acute effects of IL-6 therefore do not seem to include a stimulatory effect on GLP-1 secretion in mice.

Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas.

OBJECTIVE: Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. METHODS: The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. RESULTS: Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or ß-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. CONCLUSION: BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.

Circulating Glucagon 1-61 Regulates Blood Glucose by Increasing Insulin Secretion and Hepatic Glucose Production.

Glucagon is secreted from pancreatic α cells, and hypersecretion (hyperglucagonemia) contributes to diabetic hyperglycemia. Molecular heterogeneity in hyperglucagonemia is poorly investigated. By screening human plasma using high-resolution-proteomics, we identified several glucagon variants, among which proglucagon 1-61 (PG 1-61) appears to be the most abundant form. PG 1-61 is secreted in subjects with obesity, both before and after gastric bypass surgery, with protein and fat as the main drivers for secretion before surgery, but glucose after. Studies in hepatocytes and in ß cells demonstrated that PG 1-61 dose-dependently increases levels of cAMP, through the glucagon receptor, and increases insulin secretion and protein levels of enzymes regulating glycogenolysis and gluconeogenesis. In rats, PG 1-61 increases blood glucose and plasma insulin and decreases plasma levels of amino acids in vivo. We conclude that glucagon variants, such as PG 1-61, may contribute to glucose regulation by stimulating hepatic glucose production and insulin secretion.

Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells.

GLUTag, NCI-H716, and STC-1 are cell lines that are widely used to study mechanisms underlying secretion of glucagon-like peptide-1 (GLP-1), but the extent to which they resemble native L-cells is unknown. We used validated immunoassays for 14 different hormones to analyze peptide content (lysis samples; n = 9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n = 6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2, glicentin, and oxyntomodulin in a pattern (prohormone convertase (PC)1/3 dependent) similar to that described for human gut. All three cell lines showed basal secretion of GLP-1 and GLP-2, which increased after stimulation. In contrast to freshly isolated murine L-cells, all cell lines also expressed PC2 and secreted large amounts of pancreatic glucagon. Neurotensin and somatostatin storage was low and secretion was not consistently increased by stimulation. STC-1 cells released more glucose-dependent insulinotropic polypeptide than GLP-1 at baseline (P < 0.01) and KCl elevated its secretion (P < 0.05). Peptide YY, which normally co-localizes with GLP-1 in distal L-cells, was not detected in any of the cell lines. GLUTag and STC-1 cells also expressed vasoactive intestinal peptide, but none expressed pancreatic polypeptide or insulin. GLUTag contained and secreted large amounts of CCK, while NCI-H716 did not store this peptide and STC-1 contained low amounts. Our results show that hormone production in cell line models of the L-cell has limited similarity to the natural L-cells.