kin-29 mutants have increased total body fat and food-leaving behavior, which are partially suppressed by ATGL-1 OE.
(A) Total triglyceride (TAG) levels per μg protein. The temperature-sensitive daf-2 insulin receptor mutant was raised at 15°C (permissive) and shifted to 20°C (restrictive) at the early L4 stage before TAG measurements during the adult stage. Data are represented as the percentage of total TAG in WT controls ± SEM of 3 experiments. * and *** indicate values that are different from WT at p < 0.05 and p < 0.001, respectively, by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 4A). (B) Fat content measured with fixative Oil Red O staining for each indicated genotype. Data are represented as a percentage of body fat in WT controls ± SEM (n = 19–21 animals). *** indicates values that are different from WT at p < 0.001 by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 4B). (C) The feeding rate of kin-29 null mutants is not different from that of WT control animals. Feeding rate was measured as the number of feeding motions (pumps per minute) in the presence of food for each indicated genotype with eat-2 feeding-defective mutants showing as expected reduced feeding rate. Mean ± SEM (n = 16 animals). *** indicates values that are different from WT at p < 0.001 by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 4C). (D) ATGL-1 OE reduces the increased fat stores of kin-29 null mutants. Fat content was measured with fixative Oil Red O staining. Data are represented as a percentage of body fat in WT control animals ± SEM (n = 9–16 animals per group). ATGL-1 (OE) indicates OE of ATGL-1. *p < 0.05 and ***p < 0.001 by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 4D). (E and F) ATGL-1 OE partially restores the reduced L4 lethargus/DTS (E) and UVC-induced quiescence (F) of kin-29 null mutants. Data are represented as the mean ± SEM with n = 8–17 animals for DTS and n = 14–16 animals for SIS. ***p < 0.001 and **p < 0.01 by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 4E and 4F). (G) ATGL-1 OE reduces the increased food-leaving behavior of kin-29 null mutants. Food leaving was measured as the area of exploration with each data point representing tracks from a population outside the bacterial lawn. Each data point represents the total number of pixels outside of the bacterial lawn of 5 animals per plate, and the horizontal line represents the mean ± SEM of individual experiments (S1 Data, Sheet 4G). Representative images are shown of food-leaving behavior, in which frames from a 12-hr video were collapsed into a single image. ***p < 0.001 and **p < 0.01 by an ANOVA with Tukey multiple-comparisons test. (H) The reduced ATP levels per μg protein in L4 larvae of kin-29 null mutants are not significantly restored by ATGL-1 OE. Data are normalized to WT and represented as the mean ± SEM of 4 experiments. ***p < 0.001 by an ANOVA with Tukey multiple-comparisons test (S1 Data, Sheet 4H). (I and J) PHX treatment results in reduced body movement quiescence during L4 lethargus/DTS (I) and after UVC exposure/SIS (J) in WT animals. Total minutes of body movement quiescence during L4 lethargus, and during the first 4 hr after UVC exposure in adults in the presence (+) of 1 mM PHX or in the absence (-) of PHX. Shown is 1 representative biological replicate of an experiment performed 4 times. Data are represented as mean ± SEM with n = 9–13 animals for DTS and n = 10–19 animals for SIS. ***p < 0.001, **p < 0.0 by an unpaired 2-tailed t test (S1 Data, Sheet 4I and 4J). ATGL-1, adipose triglyceride lipase-1; DTS, developmentally timed sleep; L4 stage, fourth larval stage; ns, not significant; OE, overexpression; PHX, perhexiline; SIS, stress-induced sleep; TAG, triacylglyceride; UVC, ultraviolet C; WT, wild type.
