Activation of lipolysis in epididymal adipose tissue explants from CAV1^+/+ and CAV1^−/− mice.

<p>Adipose tissue explants were cultured from epididymal adipose tissue of CAV1^−/− and CAV1^+/+ mice and lipolysis activated using the beta-adrenergic agonist isoproterenol (Isop, 10 µM) or by direct elevation of cAMP levels using forskolin (10 µM) and IBMX (500 µM) (Fsk/IBMX). In CAV1^−/− explants neither activation regime resulted in the PKA-mediated phosphorylation of HSL on either Ser563 or Ser660, despite the expression of the PKA catalytic subunit (<b>A</b>), or stimulated the release of (<b>B</b>) glycerol or (<b>C</b>) NEFA (n = 3−5, mean ± sem).</p

Fibrosis and cell death characterises adipose tissue from CAV1^−/− mice.

<p>The release of LDH and the deposition of collagen was analysed in CAV1^+/+ and CAV1^−/− adipose tissue. (<b>A</b>) LDH activity in the culture media of epididymal adipose tissue explants after 4 h <i>in vitro</i> (mean ± sem, n = 5−6). (<b>B</b>) Release of LDH from adipose tissue explants during collagenase digestion (mean ± sem, n = 3). (<b>C</b>) Sirius red staining for collagen in paraffin-embedded sections of CAV1^+/+ and CAV1^−/− adipose tissue from fed and 24 h-fasted mice. (<b>D</b>) Protein levels of pro-collagen in CAV1^+/+ and CAV1^−/− adipose tissue from fed and 12 h fasted mice determined by Western blotting of whole tissue lysates. *p<0.05, **p<0.01.</p

Expression and phosphorylation of PLIN1a during fasting and <i>ex vivo</i> culture.

<p>The protein levels and phosphorylation of PLIN1a in epididymal adipose tissue from fed mice and following a 12 h fast (<b>A, B</b>). The expression of PLIN1a was quantified relative to actin. Phosphorylation of PLIN1a was determined using an antibody to phosphorylated PKA-substrate (RRxS/T). (<b>C, D</b>) The protein level of PLIN1a was compared between tissue (Ti) and explants of the same tissue following <i>ex vivo</i> culture for 4 h (Ex). Adipose tissue was analysed from mice maintained on a Control or high fat (HFD) diet for 12 weeks. The expression of PLIN1a in explants was quantified relative to tissue (mean ± sem, n = 3). *p<0.05, ***p<0.001.</p

Macrophage infiltration and IL-6 analysis in CAV1^−/− adipose tissue.

<p>The infiltration of macrophage into adipose tissue and the release of IL-6 was analysed in CAV1^+/+ and CAV1^−/− adipose tissue from fed and 24 h-fasted mice. (<b>A</b>) Immunohistochemistry of the macrophage marker protein F4/80 showing macrophage infiltration in CAV1^+/+ and CAV1^−/− adipose tissue from fed and 24 h-fasted mice. (<b>B</b>) Quantitation of F4/80-positive infiltrated macrophage (n = 6−8 mice). (<b>C</b>) The release of IL-6 from epididymal adipose tissue explants of CAV1^+/+ and CAV1^−/− mice was measured over an 8 h period (mean ± sem, n = 3−6, except 8 h where n = 2). (<b>D</b>) The basal release of glycerol from unstimulated explants was determined after 4 h in culture (mean ± sem, n = 5−6). (<b>E</b>) Systemic IL-6 levels in serum from CAV1^−/− and CAV1^+/+ mice following a 24 h fast (mean ± sem, n = 10−11 mice). (<b>F</b>) The release of mIL-6 over 4 h from epididymal adipose tissue explants of CAV1^+/+ and CAV1^−/− mice maintained on a Control or high fat diet (HFD) (mean ± sem, n = 5−6). *p<0.05, **p<0.01, ***p<0.001.</p

Assessment of lipid uptake in WT and <i>sg/sg</i> BMMs.

<p>(a) TLC was performed on lipid extracted from WT and <i>sg/sg</i> BMMs (n = 3 littermate pairs). Lanes 2–4 and 5–7 correspond to untreated WT and <i>sg/sg</i> samples respectively while lanes 8–10 and 11–13 correspond to 4 h 400 μM oleic acid-treated WT and <i>sg/sg</i> samples respectively. Lanes 1 and 14 are ladder standards. Quantification was performed by normalizing the relative density of triglyceride bands in each lane to WT untreated controls (arbitrarily set as 1.0) and is expressed as the mean ± S.E.M. relative fold-difference (n = 3 littermate pairs). Statistical analyses were performed using two-way ANOVAs with Bonferroni post test applied where *P<0.05. (b) Representative images of WT and <i>sg/sg</i> BMMs with endocytosed fluorescent-acLDL were captured using the Olympus upright wide-field epifluorescence microscope with actin (phalloidin; white) and nucleus (DAPI; blue) labeling, and fluorescent-acLDL (red). Quantification of fluorescent-acLDL uptake is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) normalized to number of cells analyzed (n = ~200 cells per mouse) from n = 4 biological replicates (littermate pairs). Relative fold-difference is calculated with the mean of WT values set as 1.0 and is available on the right Y-axis. Statistical analysis was performed using an unpaired Student’s t-test where **P<0.01. Scale bars = 20 μm. (c) Electron microscopy of LDs. Representative images of WT and <i>sg/sg</i> BMMs treated with 400 μM oleic acid overnight show clusters of LDs accumulated in the cytoplasm. And interspersed with endoplasmic reticulum. N = nucleus, scale bars = 1 μm. Quantification of LD size is expressed as a histogram of area/LD frequency distribution.</p

Assessment of lipid storage in WT and <i>sg/sg</i> BMMs.

<p>(a) Representative images of BMMs from WT and <i>sg/sg</i> littermates fixed and stained for LDs using Oil Red O (red). Images were taken using the Personal Deltavision Deconvolution microscope with wheat germ agglutinin (white) and nucleus (DAPI; blue) labeling. Quantification of LDs is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs normalized to number of cells analyzed (n = ~100 cells/mouse) from n = 4 littermate pairs. (b) Representative images of WT and <i>sg/sg</i> BMMs immunolabeled for ADRP (red). Images were taken using the Personal Deltavision Deconvolution microscope (maximum projection of 30-step 0.1 μm slices) with actin (white) and nucleus (DAPI; blue) labeling. Quantification of LDs was performed on 700–1300 LDs acquired from 10 fields of view per biological replicate (n = 3 littermate pairs) and is expressed as the mean ± S.E.M. top-down cross sectional area, perimeter, and Feret’s diameter (distance between two parallel tangential planes) per LD. Statistical analyses for were performed using unpaired two-tailed Student’s t-tests where *P<0.05; **P<0.01. Scale bars = 10 μm.</p

Increased <i>Vldlr</i> mRNA expression correlates with increased lipid uptake in <i>sg/sg</i> BMMs.

<p>(a) <i>Vldlr</i> mRNA expression from Lipoprotein Signaling and Cholesterol Metabolism RT² Profiler PCR Array (Qiagen) expressed as the mean ± S.E.M. relative quantification (RQ) (fold-change) in <i>si</i>Ch25h cells (black bar) compared to control siRNA (set as 1.0; white bar). The Genorm software embedded in StatMiner determined <i>Actb</i>, <i>Gapdh</i>, and <i>Hsp90ab1</i> to be the three most stable and robust endogenous controls and all data were normalized to the median of their Ct values. The P-values were calculated using empirical Bayes statistics where *P<0.05. (b) Targeted qPCR analysis was performed using Taqman Gene Expression Assays for <i>Vldlr</i>. <i>Hprt1</i> was used as the endogenous control. Analysis was performed using RNA acquired from n = 4 littermate pairs of WT and <i>sg/sg</i> BMMs in triplicate experiments and represented as the mean ± S.E.M relative quantification (fold-change) with WT set as 1.0. Statistical analysis was performed using unpaired two-tailed Student’s t-test where *P<0.05; **P<0.01. (c) RAW264.7 cells were transiently transfected with either an empty HA-tagged pcDNA3.1 vector or a vector containing HA-VLDLR for 48 h. Cells were stained for LDs using Oil Red O and quantification of LDs is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs normalized to number of cells analyzed (~350 cells per experiment, n = 3 independent experiments). Statistical analysis was performed using an unpaired two-tailed Student’s t-test where *P<0.05. Representative images of RAW264.7 cells transiently transfected with either an empty HA-tagged pcDNA3.1 vector or a vector containing HA-VLDLR for 48 h were captured using the Olympus upright wide-field epifluorescence microscope are shown in the bottom panel. Red denotes LDs (Oil Red O staining) and blue denotes nuclei (DAPI). Scale bars = 100 μm.</p

<i>Ch25h</i> knockdown up-regulates LDs.

<p>(a) Western blot analysis of Ch25h protein in WT (lanes 1 to 4) and <i>sg/sg</i> (lanes 6 to 9) BMMs (n = 4 littermate pairs). Lane 5 was only loaded with sample buffer. Quantification was performed by normalizing the relative densities of Ch25h bands (32 kDa) to the loading control ERp72 (72 kDa) and is represented as the mean ± S.E.M. relative density from n = 4 littermate pairs. Statistical analysis was performed using an unpaired two-tailed Student’s t-test where **P<0.01. (b) Two separate siRNA oligomers (individually or pooled) were used to knockdown expression of <i>Ch25h</i> in WT BMMs. The degree of mRNA knockdown was assessed by qPCR in unactivated macrophages with <i>Hprt1</i> as the housekeeping gene and represented as the mean ± S.E.M relative quantification (fold-change) with WT cells treated with siRNA negative control set as 1.0. Statistical analysis was performed using a one-way ANOVA with Bonferroni’s post test applied, comparing Ch25h mRNA expression in all treatments relative to the siRNA negative control (n = 4 biological replicates) where *P<0.01; n.s. denotes non-significant. (c) Quantification of LDs in WT control and <i>si</i>Ch25h BMMs is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs (Oil Red O) normalized to number of cells analyzed (~500–700 cells per biological replicate (n = 4)). Statistical analysis was performed using an unpaired two-tailed Student’s t-test where ***P<0.001. (d) Representative images of LDs (stained using Oil Red O) in WT control and <i>si</i>Ch25h BMMs. Images were taken using the Olympus upright wide-field epifluorescence microscope. Blue denotes nucleus (DAPI) labeling and red denotes LD (Oil Red O) labeling. Scale bars = 50 μm. (e) Representative images of anti-ADRP (red) labeling in WT control and <i>si</i>Ch25h BMMs. Images were taken using the Personal Deltavision Deconvolution microscope and displayed as the maximum projection of 30-step z-stack (0.1 μm slices). White denotes actin (phalloidin) labeling and blue denotes nucleus (DAPI) labeling. Quantification of LDs was performed on 600–700 LDs acquired from 5 fields of view per biological replicate (n = 4 biological replicates) and is expressed as the mean ± S.E.M. cross sectional area, perimeter, and Feret’s diameter per LD. Statistical analyses were performed using unpaired two-tailed Student’s t-tests for each parameter where **P<0.01. Scale bars = 10 μm. (f) <i>sg/sg</i> BMMs treated with vehicle control (0.01% DMSO) or 25HC (0.025 μM to 0.1 μM) for 4 h. Representative images were taken using the Olympus upright wide-field epifluorescence microscope are shown on the left panel where blue denotes nucleus (DAPI) labeling and red denotes LD (Oil Red O) labeling. Quantification of LDs (low 25HC treatment) is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs normalized to number of cells analyzed (n = ~100 cells per mouse per treatment). Statistical analysis was performed using a one-way ANOVA with Bonferroni’s post test (n = 3 biological replicates), comparing the amount of LDs in each treatment to the vehicle control <i>sg/sg</i> cells where **P<0.01; ***P<0.001; n.s. denotes non-significant. Scale bars = 50 μm.</p

Expression of a single Rab6 inactive isoform inhibits TNF secretion.

<p>(A) After 2 h of LPS incubation Rab6a(T27N)–GFP transfected RAW 264.7 cells do not show plasma membrane staining for TNF (asterisks), while untransfected cells (arrowheads) clearly show surface staining indicative of TNF secretion. (B) Expression of Rab6a(T27N)–GFP results in accumulation of TNF in the Golgi complex. (C) Quantification of the TNF released in the growing medium per each experimental point has been graphed. Control is untransfected cells. Original optical magnification 63X (A, B). Bar: 15 µm (A, B). *** = p<0.001 (pairwise comparisons).</p

siRNA Rab6 affects the p230 localization on the Golgi membranes which is required for TNF secretion.

<p>(A) siRNA Rab6 RAW 264.7 macrophages were co-transfected with SidC_P4C–GFP and GalT–mCherry (a), golgin-97(GRIP)–mCherry (b), p230(GRIP)–mCherry (c). Representative co-localization passing through the line scan (a-c), and plotted on the adjacent graphs (a'-c'), shows a clear decreased localization of p230 on the Golgi membranes (c'), less so efficient for golgin-97 (b'). (B) In the same experimental conditions, RAW 264.7 macrophages were stained for intracellular (a, b) and surface (a''', b''') TNF in the presence of LPS in control (a-a''') and in siRNA Rab6 (b-b''') cells. The addition of TAPI (a''', b''') was used to block TNF cleavage on plasma membrane, otherwise released into the growth medium, and to visualize the TNF staining on the surface of the cells. The depletion of Rab6 inhibits the arrival of TNF on the plasma membrane (b'''), which is concomitant with a partial redistribution of p230 (b'). Original optical magnification 63X. Bar: 10 µm (Aa-b, Ba''', Bb-b''), 15 µm (Bb'''), 20 µm (Ba–a'').</p