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

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