The excessive storage of neutral lipids in lipid droplets (LDs) is a consequence of excess dietary nutrient uptake and the primary cause of major metabolic disorders, including obesity, diabetes, and atherosclerosis (Eckel et al., 2005). Several studies investigating proteins associated with the monolayer surface of LDs have identified the Rab18 GTPase as a component with a role in the regulation of fat storage and lipid mobilization (Martin et al., 2005; Ozeki et al., 2005). Rab18, a small GTPase protein localized on the surface of LDs, plays a key role in several LD-related processes, including lipogenesis, lipolysis, and lipophagy (Pulido et al., 2011; Schulze et al., 2017). Although Rab18 has been previously associated with multiple roles in lipid metabolism, including mediating the apposition of LDs to the ER, its fundamental function remains disputed (Ozeki et al., 2005). To better understand the function of Rab18 in fat control at the organismal level, we proceeded to investigate the effect of
rab-18 knockout on overall lipid abundance in C. elegans.To determine if RAB-18 regulates fat storage in C. elegans, we first investigated the fat content of
rab-18(
ok2020) mutants compared to wild type (N2) worms by performing fixed Nile Red (NR) and Oil Red O (ORO) staining experiments under fed conditions. Each NR and ORO experiment was performed with 5 different samples of N2 and
rab-18(
ok2020) worms and 10 worms were analyzed from each sample. Three separate NR and ORO experiments were performed for a total of 150 images per strain.
rab-18 mutants displayed a significant reduction in NR fluorescence intensity (Figure 1A, B) and ORO staining intensity (Figure 1A, C) compared to the control N2 worms. This reduction suggests a significant decrease in lipid content in
rab-18(
ok2020) mutants compared to N2 worms. The less pronounced reduction in the fat content of
rab-18(
ok2020) mutants observed in ORO experiments is attributed to the lower sensitivity of ORO in quantifying changes of lipid abundance compared to NR (Escorcia et al., 2018).The fixed NR staining experiment of wild type (N2) and
rab-18(
ok2020) mutant worms was also performed under short-term (6 hours) fasting conditions to determine whether the previously observed decrease in fat content of mutant worms under fed conditions was amplified or reduced under short-term fasting conditions. Moreover, fixed NR staining under fasting conditions serves as an internal control to ensure the accuracy of the staining method as the reduction in LD fat content through lipolysis upon short-term fasting is widely acknowledged in the literature (Srinivasan et al., 2015). Three separate NR staining experiments were performed and 10-15 images were captured per strain for each experiment. Representative images are shown in Figure 1D and quantified in Figure 1E. A significant reduction in NR fluorescence intensity was observed in
rab-18(
ok2020) mutants as compared to wild type (N2) under fasted conditions, suggesting a significant decrease in lipid content. Moreover, we demonstrate a decrease in fat content under short-term (6 hours) starving conditions in both wild type and
rab-18(
ok2020) mutant worms, which is in line with findings that short-term fasting depletes body fat stores due to the increased expression of ATGL (
atgl-1) lipase mediating lipolysis (Srinivasan et al., 2015). Thus, RAB-18 promotes lipid storage under both fed and fasted conditions and is not required for lipolysis during short-term fasting.