Fig. 3
From: Methyl group donors abrogate adaptive responses to dietary restriction in C. elegans
Choline abrogates the delayed depletion of the fat stores of dietarily restricted worms. a Depletion of lipid droplets in response to starvation (24 h) in ad libitum-fed L4 larvae (a, c) and dietarily restricted L4 larvae (b, d), without (H2O vehicle control; a, b), or with choline (c, d) supplementation. Lipid droplets were visualized after short-term (20 min) vital BODIPY™ 493/503 staining using a ×63 objective and Zeiss Apotome equipment. Image stacks with a depth of 10 μm, consisting of 21 focal planes at 0.5-μm intervals, were captured and rendered as maximum projections. The images show intestinal lipid droplets in the mid-regions of the worms. The BODIPY™ 493/503 fluorescence signals (green) and the auto-fluorescence signals of lysosome-related organelles (red) are shown to study possible co-localizations of the signals. Scale bars, 20 and 10 μm (magnifications). b–e Quantification of intestinal lipid droplets using the 3D spot segmentation plug-in of ImageJ software. The lipid droplets of ad libitum-fed (AL) and dietarily restricted (DR) L4 larvae were analyzed at time point T0 (0 h, no starvation) and in response to starvation for 24 or 72 h. The distributions of the relative lipid droplet number/fraction (b) and the relative lipid droplet volume/fraction (c) per lipid droplet size category (<1, 1–3, 3–5, 5–10, >10 μm3) are shown. Table d shows the relative lipid droplet volume in response to 72 h of starvation (% of T0) among different lipid droplet size categories (>10, 5–10, >5 μm3). e shows the summarized LD volume/area (AU, arbitrary unit), a proxy indicator of the extent of fat storage. The data were derived from six to nine individual worms per feeding condition in three independent experiments. f, g Triacylglyceride (TAG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) contents were assessed using thin-layer chromatography (TLC). The lipid contents of ad libitum-fed (AL) and dietarily restricted (DR) L4 larvae were analyzed at time point T0 (0 h, no starvation) and in response to starvation for 24 or 72 h. Table g shows the relative ratio of the TAG and phospholipid (PL; PE+PC) contents in response to 72 h of starvation (% of T0). These ratios correspond to the lipid droplet volume/area. Five-microliter samples containing lipids from equal numbers of worms (166 or 333) were dotted onto each lane to ensure that the amounts of triacylglycerides and phospholipids were in the linear range of the triolein and phospholipid standard curve, respectively. The assays were conducted in duplicate, and six to eight independent experiments are summarized. AL ad libitum, DR dietary restriction, DR CH dietary restriction condition supplemented with choline