CRT N-terminal fragments are re-localized at the cell surface.

<p>(<b>A</b>) The kinetics of the appearance of the F protein and CRT C-terminal and N-terminal fragments at the surface of infected Vero cells were monitored by flow cytofluorimetry using the corresponding specific antibodies. Control cell cultures were not infected (grey lines). From infected cell cultures, infected cells (red line; GFP-positive) and non-infected cells (blue line, GFP-negative) were identified by virus-encoded GFP fluorescence. Cell surface immuno-labelling of cells (unifxed and nonpermeablized) with F and CRT C-terminal and N-terminal were performed with specific antibodies as indicated at the top of the panels. The mean fluorescence intensity (MFI) of the labelling was determined within each cell population regularly over 48 hours of infection (top panels). The three bottom panels represent the distribution of the MFI within each population at 48 hours post-infection. Here is shown one representative experiment out of three independent experiments. (<b>B</b>) Membrane localisation of CRT N-terminal fragment following CDV infection. At 24 hours post-infection in Vero cells, cultures were immuno-labelled for the viral F protein to identify infected cells (Ba; fluorochrome: FITC), C-terminal specific anti-CRT antibody (Bb; fluorochrome: CY3), and N-terminal specific anti-CRT antibody (Bc; fluorochrome: CY5). In panel Bd, cell membranes were stained with alexa-405-conjugated wheat germ agglutinin (WGA). The merges images b and d (Be) reveal little co-localisation of CRT C-terminal fragment with the cell surface, while the merges images c and d (Bf) indicate partial surface localization of the CRT N-terminal fragment. Panel g is a merge between images b and c. Scale bar, 30 µm. Immunofluorescence analyses were performed in fixed and permeabilized cells.</p

DataSheet_1_Membrane estrogen receptor-α contributes to female protection against high-fat diet-induced metabolic disorders.pdf

BackgroundEstrogen Receptor α (ERα) is a significant modulator of energy balance and lipid/glucose metabolisms. Beyond the classical nuclear actions of the receptor, rapid activation of intracellular signaling pathways is mediated by a sub-fraction of ERα localized to the plasma membrane, known as Membrane Initiated Steroid Signaling (MISS). However, whether membrane ERα is involved in the protective metabolic actions of endogenous estrogens in conditions of nutritional challenge, and thus contributes to sex differences in the susceptibility to metabolic diseases, remains to be clarified.MethodsMale and female C451A-ERα mice, harboring a point mutation which results in the abolition of membrane localization and MISS-related effects of the receptor, and their wild-type littermates (WT-ERα) were maintained on a normal chow diet (NCD) or fed a high-fat diet (HFD). Body weight gain, body composition and glucose tolerance were monitored. Insulin sensitivity and energy balance regulation were further investigated in HFD-fed female mice.ResultsC451A-ERα genotype had no influence on body weight gain, adipose tissue accumulation and glucose tolerance in NCD-fed mice of both sexes followed up to 7 months of age, nor male mice fed a HFD for 12 weeks. In contrast, compared to WT-ERα littermates, HFD-fed C451A-ERα female mice exhibited: 1) accelerated fat mass accumulation, liver steatosis and impaired glucose tolerance; 2) whole-body insulin resistance, assessed by hyperinsulinemic-euglycemic clamps, and altered insulin-induced signaling in skeletal muscle and liver; 3) significant decrease in energy expenditure associated with histological and functional abnormalities of brown adipose tissue and a defect in thermogenesis regulation in response to cold exposure.ConclusionBesides the well-characterized role of ERα nuclear actions, membrane-initiated ERα extra-nuclear signaling contributes to female, but not to male, protection against HFD-induced obesity and associated metabolic disorders in mouse.</p

Mechanistic model of neurodegenerative processes induced by CDV infection.

<p>The F and H CDV proteins are accumulating in the ER. This event induces an early ER stress event. In early ER stress, the quantities of CRT chaperon increase, the Ca²⁺ homeostasis is altered and Ca²⁺ is depleted from ER stores. Increase of cytosolic Ca²⁺ can have as consequence a glutamate release during CDV infection as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032803#pone.0032803-Brunner1" target="_blank">[3]</a>. Glutamate release could induce, in the neighbouring neurons, Ca²⁺ entry followed by an ER stress induction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032803#pone.0032803-Chen1" target="_blank">[56]</a>. During ER stress, the infected cells show enhance expression of the chaperons CRT, calnexin and GRP94 and relocalisation of the transcription factor ATF-6 in the nucleus followed by the expression of the proapoptotic factor CHOP/GADD 153. More importantly, infected cells show CRT fragmentation in a CDV-dependent manner. C-terminal fragments are retained in the ER by the KDEL signal whereas CRT N-terminal fragments are present after 24 hours at the cell surface. Cell surface exposition of CRT N-terminal fragment may contribute to CDV-mediated neurodegenerative auto-immunity. In <b><i>grey italic</i></b> are events described in previous publications.</p

CDV infection of Vero cells causes CRT fragmentation with vasostatin formation.

<p>(<b>A</b>) Schematic representation of the 60 kDa CRT protein. The globular 27 kDa N-terminal domain (N-term) is the most important antigenic site of the protein. This domain, as well as the P domain, possesses the chaperon function. C-terminal domain (C-term) is important for Ca²⁺ storage and possesses the KDEL ER retention signal. P and C-terminal domains have together an estimated mass of 30 kDa. (<b>B</b>) The Vero cells were either left non-infected or infected with CDV. At 48 h post-infection cells were lysed and analyzed by Western blot using C-terminal-specific (left) or N-terminal-specific (right) antibodies. Note the C-terminal 30 kDa fragment, and the 27 kDa N-terminal fragment. GAPDH is used as an internal control. (<b>C</b>) Impact of the ER stress inducing drugs dithiothreitol (DTT) and thapsigargin (Th) on CRT expression during CDV infection. Red fluorescence in all panels corresponds to CRT immunostaining, which increases in a DTT concentration-dependent manner (top panels) or in a thapsigargin concentration-dependent manner (white arrow heads). For comparison, calreticulin staining is shown in infected cells probed by immunostaining of the F protein (bottom left and insert panels). Immunofluorescence analyses were performed in fixed and permeabilized cells. (<b>D</b>) Western blot using the antibody recognizing either the N- terminal domain of CRT (27 kDa, right panel) or the C- terminal domain (30 kDa; left panel). Both antibodies recognize the full-length CRT (top line, 60 kDa). Cellular extracts come from Vero cells exposed to DTT or thapsigargin (Th), or CDV infected as indicated. GAPDH was used as an internal control. CRT cleavage was specifically mediated by CDV infection (line 11) and not by exposure to DTT or thapsigargin.</p

Infection of Vero cells by rgA75/17-V (CDV) induces ER stress.

<p>(<b>A, B, C, D</b>) Representative photomicrographs of non-infected Vero cells (A) and infected with a Vero cell-adapted canine distemper virus (CDV) strain (rgA75/17-V). The former recombinant CDV expresses the enhanced green fluorescence protein (e-GFP) for easier identification of infected cells (B, C, D). Cultures were infected 1 day after seeding. Cells were then fixed and permeabilized and subsequently analysed by immunofluorescence at 24 hours (A, B and D) or 48 hours (C) after infection. Antibodies against the protein F of CDV (F), calreticulin, CHOP-GADD and Calnexin are as indicated in the panels. Merged images are shown on bottom panels, including labelling with 4′6-diamidino-2-phenylindole (DAPI, blue). Scale bar, 30 µm. Calreticulin and calnexin expression are increased in infected cells that expressed the F protein (B and D) and at 48 hours post-infection, infected cells also express strongly the nuclear proapoptotic CHOP/GADD 153 (C). (<b>E</b>) Increase of CRT, CHOP/GADD 153 and calnexin during culture infection, as determined by flow cytofluorimetry. Each sample was analysed in triplicate on three separate experiments, and one representative experiment is shown here.</p

LXR protects from hepatic damage induced by an oleic-rich diet.

<p>(A) <i>Tnf</i>α, <i>Ccl2</i>, <i>F4/80</i>, <i>Cd68</i> and <i>Il1β</i> mRNA quantification assayed by qPCR. (B) Plasma activity of ALT and AST. (C) Plasma cholesterol and lathosterol levels analyzed by gas chromatography. Data are the mean±SEM of values measured in LXR+/+ and LXR-/- mice fed the REF or the OLIV diet. ^a Significant genotype effect. ^b Significant difference versus REF diet (n = 6 mice per group).</p

Effects of an oleic acid-rich diet on hepatic fatty acid profile in LXR+/+ and LXR-/- mice.

<p>Effects of an oleic acid-rich diet on hepatic fatty acid profile in LXR+/+ and LXR-/- mice.</p

High oleic acid diet modulates hepatic gene expression.

<p>Hepatic gene expression of 142 genes related to lipid metabolism, nuclear receptor signaling and inflammation were quantified by qPCR from liver of LXR+/+ and LXR-/- mice fed the REF or the OLIV diet. Data are presented as a heatmap associated with a hierarchical classification.</p

LXR mediate the induction of lipogenesis induced by an oleic acid-rich diet.

<p>(A) Hepatic <i>Acly</i>, <i>Acaca</i>, <i>Acacb</i>, <i>Fasn</i>, <i>Elovl6</i>, <i>Scd1</i> mRNA levels quantified by qPCR. (B) Cytoplasmic protein expression levels of P-ACLY, ACLY, ACC, ELOVL6, SCD1, FASN AND β-ACTIN assayed by Western Blotting. (C) <i>Fads1</i>, <i>Fads2</i>, <i>Elovl5</i>, <i>Gpat</i>, <i>Pnpla3</i> and <i>Lpk</i> mRNA quantification assayed by qPCR. (D) <i>Srebp-1c</i> and <i>Chrebp</i> mRNA quantification assayed by qPCR. (E) Cytoplasmic and nuclear expression levels of LXR, SREBP-1c and ChREBP assayed by Western Blotting. Data are the mean±SEM of values measured in LXR+/+ and LXR-/- mice fed REF or OLIV diet. ^a Significant genotype effect. ^b Significant difference versus REF diet (n = 6 mice per group).</p

High oleic acid diet induces hepatic steatosis in LXR+/+ but not in LXR-/- mice.

<p>(A) Representative Oil Red O-stained frozen sections of liver from mice of both genotypes fed the REF or the OLIV diet (Scale bars: 100 μm). Neutral lipids appear in red. (B) Liver triglycerides, cholesterol and cholesterol esters measured by gas chromatography. Data are the mean±SEM of values measured in LXR+/+ and LXR-/- mice fed the REF or the OLIV diet. ^a Significant genotype effect. ^b Significant difference versus REF diet (n = 6 mice per group).</p