Whole cell lipidome of control and LB-rich RBL2H3.

<p>RBL2H3 were grown for 6d with insulin-FDI. Individual major lipid species were separated by high performance liquid chromatography (HPLC) and fatty acid methyl esters (FAME) from each class were produced and subsequently analyzed by GC/MS. <b>A.</b> The total fold change in nanomolar amounts of each fatty acid within each class of lipids are presented in a heatmap using RColorBrewer and gplots in R. Negative fold change moves towards purple whereas positive fold change moves toward green. Trace lines are used to reinforce change within the groups and are relative to a dashed median line. The density of change is tracked within the scale bar to the left. <b>B.</b> Change in absolute TAG and FFA levels summed between untreated and insulin-treated cell samples. <b>C</b>. The average fold change of fatty acids between the two conditions is bar plotted and organized by the degree of saturation (saturated, mono-unsaturated (MUFA), and poly-unsaturated (PUFA)) of the fatty acids. The legend on the right side indicates the scale in fold change from -3.5 to 6. <b>D.</b> Individual FA in the AA biosynthetic pathway were quantified according to their respective major lipid class. Fold change was calculated based on response to insulin-FDI in treated compared to control mast cells (18:2n6, linoleic acid; 18:3n6, linolenic acid; 20:3n6, di-homo-gamma-linolenic acid; 20:4n6, arachidonic acid). <b>E.</b> Experiment as in <b>D</b>, with quantification of individual FA directly involved in the AA biosynthesis pathway quantified by lipid class in terms of absolute concentration (nmol of lipid per billion cells). <i>Cholesterol Ester (CE)</i>, <i>Cardiolipin (CL)</i>, <i>Triacylglycerol (TAG)</i>, <i>Diacylglycerol (DAG)</i>, <i>Free Fatty Acid (FFA)</i>, <i>Phosphatidylserine (PS)</i>, <i>Phosphatidylcholine (PC)</i>, <i>Phosphatidylethanolamide (PE)</i>, <i>Lysophoshatidylcholine (LYPC)</i>.</p

Chronic Insulin Exposure Induces ER Stress and Lipid Body Accumulation in Mast Cells at the Expense of Their Secretory Degranulation Response

<div><p>Lipid bodies (LB) are reservoirs of precursors to inflammatory lipid mediators in immunocytes, including mast cells. LB numbers are dynamic, increasing dramatically under conditions of immunological challenge. We have previously shown <i>in vitro</i> that insulin-influenced lipogenic pathways induce LB biogenesis in mast cells, with their numbers attaining steatosis-like levels. Here, we demonstrate that <i>in vivo</i> hyperinsulinemia resulting from high fat diet is associated with LB accumulation in murine mast cells and basophils. We characterize the lipidome of purified insulin-induced LB, and the shifts in the whole cell lipid landscape in LB that are associated with their accumulation, in both model (RBL2H3) and primary mast cells. Lipidomic analysis suggests a gain of function associated with LB accumulation, in terms of elevated levels of eicosanoid precursors that translate to enhanced antigen-induced LTC4 release. Loss-of-function in terms of a suppressed degranulation response was also associated with LB accumulation, as were ER reprogramming and ER stress, analogous to observations in the obese hepatocyte and adipocyte. Taken together, these data suggest that chronic insulin elevation drives mast cell LB enrichment <i>in vitro</i> and <i>in vivo</i>, with associated effects on the cellular lipidome, ER status and pro-inflammatory responses.</p></div

ER distension and lipid biogenesis in insulin-exposed RBL2H3.

<p><b>A. Electron micrographs of control and insulin-FDI exposed RBL2H3 showing normal (left) and distended (right) ER.</b> Images are digitally zoomed from 5000x original plates. <b>B. Area analysis of ER by electron and fluorescence microscopy</b>. Cytoplasmic (n of 20 per condition) ROI were drawn on either micrographs (Image J) or confocal images of cells stained with ER-Tracker dye (NIS Elements). Data are expressed as percentage area of ROI occupied by ER. <b>C. Confirmation of ER enrichment in purified ER/microsomal fractions.</b> ER/microsomal fractions were prepared by ultracentrifugation as described in Methods and Western blotted for enrichment in the ER-resident chaperone Calnexin. Relative band intensities are shown on each panel (Image J). <b>D. Comparison of protein and lipid levels in ER/microsomal fractions prepared from control and Insulin-FDI treated RBL2H3</b>. Total protein was assessed by BCA analysis, and total lipid content was assessed by ORO absorbance assay after Bligh-Dyer extraction. <b>E. Characterization of ER Fatty acids</b>. RBL2H3 were grown for 6d with insulin-FDI as described. Isolated ER was analyzed by GC/MS and variations in ER lipids versus control levels were organized by fold change (values along y-axis) and abundance. <i>Green</i>, > 2 fold increase in treated over controls; <i>gray</i>, no change; <i>red</i>, decrease to < 50% of control levels. <b>F. Relative abundance of eicosatrienoic, arachidonic and linoleic acid in ER from control and Insulin-FDI treated cells</b>. <b>G. Summary of alterations in saturated (SFA), mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty acids in ER/microsomal fractions from control and insulin FDI-treated cells</b>.</p

Whole cell lipidome of control and LB-rich RBL2H3.

<p>RBL2H3 were grown for 6d with insulin-FDI. Individual major lipid species were separated by high performance liquid chromatography (HPLC) and fatty acid methyl esters (FAME) from each class were produced and subsequently analyzed by GC/MS. <b>A.</b> The total fold change in nanomolar amounts of each fatty acid within each class of lipids are presented in a heatmap using RColorBrewer and gplots in R. Negative fold change moves towards purple whereas positive fold change moves toward green. Trace lines are used to reinforce change within the groups and are relative to a dashed median line. The density of change is tracked within the scale bar to the left. <b>B.</b> Change in absolute TAG and FFA levels summed between untreated and insulin-treated cell samples. <b>C</b>. The average fold change of fatty acids between the two conditions is bar plotted and organized by the degree of saturation (saturated, mono-unsaturated (MUFA), and poly-unsaturated (PUFA)) of the fatty acids. The legend on the right side indicates the scale in fold change from -3.5 to 6. <b>D.</b> Individual FA in the AA biosynthetic pathway were quantified according to their respective major lipid class. Fold change was calculated based on response to insulin-FDI in treated compared to control mast cells (18:2n6, linoleic acid; 18:3n6, linolenic acid; 20:3n6, di-homo-gamma-linolenic acid; 20:4n6, arachidonic acid). <b>E.</b> Experiment as in <b>D</b>, with quantification of individual FA directly involved in the AA biosynthesis pathway quantified by lipid class in terms of absolute concentration (nmol of lipid per billion cells). <i>Cholesterol Ester (CE)</i>, <i>Cardiolipin (CL)</i>, <i>Triacylglycerol (TAG)</i>, <i>Diacylglycerol (DAG)</i>, <i>Free Fatty Acid (FFA)</i>, <i>Phosphatidylserine (PS)</i>, <i>Phosphatidylcholine (PC)</i>, <i>Phosphatidylethanolamide (PE)</i>, <i>Lysophoshatidylcholine (LYPC)</i>.</p

ER reprogramming, ER stress and autophagy in insulin-treated RBL2H3.

<p><b>A, B. Altered expression of markers of ER stress, the UPR and autophagy.</b> RBL2H3 were treated with insulin-FDI for 6d and protein lysates were prepared. Western blot analysis (antibody concentrations indicated in micrograms/ml)was performed using UPR markers anti-IRE1 alpha (0.5), anti-phospho PERK-Thr980 (0.1), anti-ATF6 (2.5) and loading control anti-GRB2 (0.05)(A) and autophagy markers (B) anti-ATG3 (0.5), anti-ATG12 (0.5), anti-ATG7 (0.5), anti-Beclin (0.5), anti-LC3A (0.1), and anti-LC3B (0.5) with anti-Grb2 as a loading control. <b>C-E.</b> Immunofluorescent identification and quantification of autophagy positive mast cells. Three markers of autophagy (Beclin-1, LC3B and ATG7) were used to quantify the percent of cells staining positively for autophagy (<b>C</b>). <b>D, E</b>. Quantification of autophagy marker immunostaining. Counting was performed in a sample-blinded fashion and expressed as % of 200 counted cells (<b>D</b>) and mean of the number of immunodecorated structures per cell (<b>E</b>).</p

Lipidome of lipid bodies isolated from insulin-exposed RBL2H3.

<p>RBL2H3 were grown for 6d with insulin-FDI and LB were isolated by ultracentrifugation. <b>A. Confirmation of LB enrichment in ultracentrifugation fractions</b>. Fractions were Western blotted for the enriched presence of the LB-associated protein perilipin A and the diminished representation of the ER-resident chaperone calnexin. Each lane resolves 10 μl of whole cell or LB protein determined by BCA assay. Inset numbers represent ratio of expression determined by integrated density of the Western band (Image J). <b>B. Lipid profile of isolated LB</b>. Individual major lipid classes were separated by high performance liquid chromatography (HPLC) and fatty acid methyl esters from each class were produced and subsequently analyzed by GC/MS. Abundance of lipid species in LB from insulin-FDI treated mast cells were quantified and organized into ribbon plots using Circos. All lipid species and nmole percentage representations of the observed classes are visualized. The Circos plots draw ribbons from the fatty acids to the different associated classes. Line width is proportional to the recorded percentage. The outer ring is representative of the total nmole percentage of either the fatty acid and/or class. The inner ring is the relative amount of each element in the plot. The values listed on the inner ring are 100x larger than the percentage in order to resolve less common fatty acids. The three different plots reduce the information from the total fatty acids (top) to the unsaturated fatty acids (middle) to arachidonic acid and its precursors (bottom). This Fig resolves which lipid class and at what percentage each arachidonic acid pathway member occurs.</p