Development of a conditionally immortalized human pancreatic β cell line

International audienceDiabetic patients exhibit a reduction in β cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human β cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human β cell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult β cells, including expression of β cell markers and insulin secretion following glucose stimulation; however, unlike primary β cells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human β cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of β cell–specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human β cells and should be a valuable tool for further analysis of human β cells

Modulatory role of PYY in transport and metabolism of cholesterol in intestinal epithelial cells.

BACKGROUND: Gastrointestinal peptides are involved in modulating appetite. Other biological functions attributed to them include the regulation of lipid homeostasis. However, data concerning PYY remain fragmentary. The objectives of the study were: (i) To determine the effect of PYY on intestinal transport and synthesis of cholesterol, the biogenesis of apolipoproteins (apos) and assembly of lipoproteins and (ii) To analyze whether the effects of PYY are similar according to whether cells are exposed to PYY on apical or basolateral surface. METHODOLOGY/PRINCIPAL FINDINGS: Caco-2/15 cells were incubated with PYY (1-36) administered either to the apical or basolateral medium, at concentrations of 50 or 200 nM for 24 hours. De novo synthesis of cholesterol, cholesterol uptake, and assembly of lipoproteins were evaluated through the incorporation of [(14)C]-acetate, [(14)C]-cholesterol, and [(14)C]-oleate, respectively. Biogenesis of apos (A-I, A-IV, E, B-48 and B-100) was examined by the incorporation of [(35)S]-methionine. The influence of PYY on protein and mRNA levels of many key mediators of lipid metabolism was analyzed by Western blot and PCR, respectively. Our results show that PYY influenced cholesterol metabolism in Caco-2/15 cells depending on the site of PYY delivery. Apical addition of PYY significantly lowered the incorporation of [(14)C]-cholesterol likely via the reduction of NPC1L1, stimulated intracellular cholesterol synthesis probably through an increase in SREBP-2 expression, whereas it concomitantly increased apo A-I synthesis and decreased LDL secretion. In contrast, basolateral PYY reduced the production of chylomicrons (CM) as well as the biogenesis of apos B-48 and B-100, while lowering the expression of the transcription factors RXRα and PPAR(α,β). CONCLUSIONS/SIGNIFICANCE: PYY is capable of influencing cholesterol homeostasis in intestinal Caco-2/15 cells depending on the site delivery. Apical PYY was able to decrease cholesterol uptake via NPC1L1 downregulation, whereas basolateral PYY diminished CM output through the biogenesis decline of apos B-48 and B-100

In Vivo Imaging of Prostate Cancer Tumors and Metastasis Using Non-Specific Fluorescent Nanoparticles in Mice

With the growing interest in the use of nanoparticles (NPs) in nanomedicine, there is a crucial need for imaging and targeted therapies to determine NP distribution in the body after systemic administration, and to achieve strong accumulation in tumors with low background in other tissues. Accumulation of NPs in tumors results from different mechanisms, and appears extremely heterogeneous in mice models and rather limited in humans. Developing new tumor models in mice, with their low spontaneous NP accumulation, is thus necessary for screening imaging probes and for testing new targeting strategies. In the present work, accumulation of LipImageTM 815, a non-specific nanosized fluorescent imaging agent, was compared in subcutaneous, orthotopic and metastatic tumors of RM1 cells (murine prostate cancer cell line) by in vivo and ex vivo fluorescence imaging techniques. LipImageTM 815 mainly accumulated in liver at 24 h but also in orthotopic tumors. Limited accumulation occurred in subcutaneous tumors, and very low fluorescence was detected in metastasis. Altogether, these different tumor models in mice offered a wide range of NP accumulation levels, and a panel of in vivo models that may be useful to further challenge NP targeting properties

Variables associated with low, moderate and high emergency department use among patients with substance-related disorders

Aims: This study identified factors associated with frequency of emergency department (ED) use for medical reasons among patients with substance-related disorders (SRD) in Quebec (Canada) for 2014-15. Methods: Participants (n = 4731) were categorized as: 1) low (1 visit/year), 2) moderate (2 visits/year), and 3) high (3+ visits/year) ED users. Independent variables included predisposing, enabling and needs factors based on the Andersen Behavioral Model. Multinomial logistic regression identified associated variables. Results: Factors positively associated with moderate and high ED use included adjustment disorders, suicidal behavior, alcohol-induced disorders, less urgent to non-urgent illness acuity, referral to local health community services centers (LHCSC) at discharge, and living in a materially deprived area. Factors positively associated with high ED use only included anxiety disorders, alcohol use disorders, drug use disorders, chronic physical illness, subacute problems, prior ED use for MD and/or SRD, prior LHCSC medical interventions, physician consultation within one month after discharge, living in very deprived or middle-class areas, and, negatively, being hospitalized for medical reasons in second ED visit. Moderate ED use only was negatively associated with alcohol intoxication and being referred to a GP at ED discharge. Conclusions: Compared to low ED users, most high users with SRD were men presenting more complex and severe conditions. They visited ED mainly for subacute or non-urgent problems. Compared to low ED users, most moderate users had alcohol-induced disorders, less alcohol intoxication, and acute common MD. They visited ED mainly for non-urgent care. Diverse strategies should be implemented to reduce ED visits, targeting each group.</p

Influence of oleic acid on PYY secretion in the apical and basolateral media.

<p>Oleic acid at the concentrations of 0.7 mM and 1.4 mM was added either to the apical or basolateral medium. Following the 2-h incubation period, both apical and basolateral media were assessed for their PYY content. Values are expressed as means ± SD for n = 3 separate experiments in each group. CTR, Control; Baso, basolateral; OA, Oleic Acid.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the synthesis of apolipoproteins (apo).

<p>Epithelial cells at 14 days post-confluence were incubated with [³⁵S]-methionine in the presence of PYY (1–36) (50 or 200 nM) and unlabeled oleic acid for 24 h to stimulate apo biogenesis. At the end of the labelling period, cells were washed, homogenized, and centrifuged. Supernatants from the cell homogenates were then reacted with excess antibodies for 18 h at 4°C to precipitate specific apos. Immune complexes were washed and analyzed by linear 4–15% SDS-PAGE. After electrophoresis, gels were sliced and counted for radioactivity. Data represent means ± SD for n = 3 separate experiments in each group. Values are reported as % of control values representing 100%. *P<0.05 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of ABCG5 and ABCG8.

<p>Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040992#pone-0040992-g002" target="_blank">Figure 2</a>. Western blot was used to analyse the protein expression of ABCG5 (A) and ABCG8 (B). Data represent means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on PPAR and SREBP-2 gene expression in Caco-2/15 cells.

<p>PCR analysis was performed on Caco-2/15 cell line at 14 days post-confluence to analyze mRNA of PPARα (A), PPARβ (B), PPARγ (C), SREBP-2 (D). Values represent means ± SD for n = 3 separate experiments in each group and are reported as percent difference relative to control. * P<0.05 vs. controls, **P<0.01 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on cholesterol synthesis.

<p>After 24 h incubation with [¹⁴C]-acetate, cells were homogenized. Lipids were extracted in chloroform/methanol and separated by TLC. The free cholesterol (FC) and cholesteryl ester (CE) bands were scraped off the plate and counted. Data represent means ± SD for n = 3 separate experiments in each group. *P<0.05 vs. controls, **P<0.01 vs. controls.</p

Effects of the administration of PYY (1–36) to the apical or basolateral medium on the protein expression of LDL-receptor (LDL-R).

<p>Caco-2/15 cells were cultured for 24 h in MEM as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040992#pone-0040992-g002" target="_blank">Figure 2</a>. Western blot was used to analyse the protein expression of LDL-R. Data represented means ± SD for n = 3 separate experiments in each group and are reported as % of control values representing 100%.</p