Fig. 1

Choline and methionine supplementation abrogates the enlargement of intestinal lipid droplets and increase in fat content observed in dietarily restricted worms. a Lipid droplets in the mid-region of ad libitum-fed L4 larvae (a, c, e, g, i, k) and dietarily restricted L4 larvae (b, d, f, h, j, l), without (H₂O vehicle control; a, b, g, h) or with choline (c, d, i, j) or methionine (e, f, k, l) supplementation, 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 planes at 0.5-μm intervals, were captured and rendered as maximal projections (a–f). Images a–f show intestinal lipid droplets. In g–l, only the first six to eight images of the respective stacks are projected to demonstrate the lipid droplets in the upper hypodermal segment. 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–f Images of intestinal lipid droplets were analyzed using the 3D spot segmentation plug-in of ImageJ software. The mean LD volume (b), maximum LD volume (c), and total LD volume are shown. The distributions of the relative number of LDs (d) and relative LD volume (e) per LD size category (<1, 1–3, 3–5, 5–10, and >10 μm³) are shown. f The summarized LD volume per area (AU, arbitrary units), a proxy indicator of the extent of fat storage, is shown (f). The data were derived from six to nine individual worms per feeding condition in three independent experiments. g, h The triacylglyceride (TAG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) contents of L4 larvae were assessed using thin-layer chromatography (TLC). g TAG content was normalized to the TOF value (time of flight), a proxy indicator of the worm’s body length. 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 the results of six to eight independent experiments are summarized. AL ad libitum, AL CH ad libitum condition with choline supplementation, DR dietary restriction, DR CH dietary restriction condition with choline supplementation, TOF time of flight, a proxy measurement of the length of the worms