Dopamine signaling regulates fat content through β-oxidation in Caenorhabditis elegans

Abstract

The regulation of energy balance involves an intricate interplay between neural mechanisms that respond to internal and external cues of energy demand and food availability. Compelling data have implicated the neurotransmitter dopamine as an important part of body weight regulation. However, the precise mechanisms through which dopamine regulates energy homeostasis remain poorly understood. Here, we investigate mechanisms through which dopamine modulates energy storage. We showed that dopamine signaling regulates fat reservoirs in Caenorhabditis elegans. We found that the fat reducing effects of dopamine were dependent on dopaminergic receptors and a set of fat oxidation enzymes. Our findings reveal an ancient role for dopaminergic regulation of fat and suggest that dopamine signaling elicits this outcome through cascades that ultimately mobilize peripheral fat depots.

Figure 1. Dopamine reduces fat content of wild type animals.

(A–D) Animals exposed to increasing concentrations of dopamine exhibit reduced fat stores as visualized by vital dyes Nile Red and BODIPY-labeled fatty acids, fixative-based dyes Oil-Red-O and Sudan Black and CARS microscopy. (A) Representative images and (C) quantitation of fluorescence intensities. Black and gray bars represent Nile Red and BODIPY respectively. (B) Illustrative images and (D) quantitation of stain intensities. Black and white bars represent Oil-Red-O and Sudan Black respectively. (E) Representative images and (F) quantification of CARS signal intensities. (C–D) Data are showed as percentage of control (“No dopamine”) average ± SEM. (n = 12 to 15 animals per condition for Nile Red and BODIPY. n = 5 to 8 animals per condition for Oil-Red-O and Sudan Black). (F) Data are showed as absolute intensity ± SD. (n = 8 to 10 worms per condition). * p<0.05, ** p<0.01, *** p<0.001 compared to “No dopamine” animals.

Figure 2. Dopamine signaling effects on fat depends on dopamine receptors.

(A–B) Fat levels of various mutants with or without exposure to dopamine. Black and white bars represent vehicle and 5 mM dopamine treated animals, respectively. Data are shown as percentage of wild type vehicle-treated average ± SEM. (n = 10–20 animals per condition per genotype). *** p<0.001 compared to vehicle-treated animals of the same genotype, ### p<0.001 compared to vehicle-treated wild type animals, +++ p<0.001 compared to dopamine-treated wild type animals.

Figure 3. Fat reducing effect of dopamine treatment is not due to behavioral changes.

(A) Pharyngeal pumping rate of wild type animals exposed to dopamine is similar to that of vehicle-treated animals over time. Data are presented as an average of 15 animals per condition per time ± SEM. (•) No dopamine, (◊) Dopamine 5 mM, (Δ) Dopamine 30 mM. (B) Dopamine causes a transient reduction in movement of wild type animals. Data are showed as the average of 5 to 9 animals per condition per time ± SEM. (•) No dopamine, (Δ) Dopamine 5 mM. ** p<0.01 compared to “No dopamine” control animals. (C) Defecation rate of dopamine-treated animals is similar to vehicle-treated animals. Data are presented as the average of 10 animals per condition ± SEM. (D–E) Chronic dopamine exposure does not affect progeny production in wild type animals. (D) Number of eggs in uterus or (E) laid per hour. Data are shown as average of nine animals per condition ± SEM.

Figure 4. Dopamine induced fat reduction requires β-oxidation machinery.

(A–B) Dopamine induced fat reduction is reduced by RNAi mediated knockdown of β-oxidation enzymes. (A) Representative images and (B) quantification of wild type animals fed with the indicated RNAi clones. Black and white bars represent vehicle and 5 mM dopamine treated animals, respectively. (C) Fluorescence quantification of 5 mM dopamine treated mutant worms available to corresponding RNAi gene clones. Data are showed as percentage of “No dopamine” control average ± SEM. (n = 8–10 animals per condition) (B) ** p<0.01 and *** p<0.001 compared to dopamine-treated animals fed on empty vector. (C) ** p<0.01 compared to “No dopamine” control animals of the same genotype. (D) Change in transcriptional levels of indicated metabolic genes upon 5 mM dopamine exposure quantified by qPCR. Dopamine increased kat-1 transcriptional level. Data are presented as average of three independent populations ± SD. Ctd – Ct dopamine exposed worms/Ctc – Ct No dopamine worms. * p<0.05 compared to “No dopamine” control animals. (E) Dopamine-treated animals exhibit an increase in β-oxidation rate. Data are presented as average of 12 independent populations ± SEM. * p<0.05 compared to “No dopamine” control animals.