Neural and molecular dissection of a C. elegans sensory circuit that regulates fat and feeding

Abstract

A major challenge in understanding energy balance is deciphering the neural and molecular circuits that govern behavioral, physiological, and metabolic responses of animals to fluctuating environmental conditions. The neurally expressed TGF-beta ligand DAF-7 functions as a gauge of environmental conditions to modulate energy balance in C. elegans. We show that daf-7 signaling regulates fat metabolism and feeding behavior through a compact neural circuit that allows for integration of multiple inputs and the flexibility for differential regulation of outputs. In daf-7 mutants, perception of depleting food resources causes fat accumulation despite reduced feeding rate. This fat accumulation is mediated, in part, through neural metabotropic glutamate signaling and upregulation of peripheral endogenous biosynthetic pathways that direct energetic resources into fat reservoirs. Thus, neural perception of adverse environmental conditions can promote fat accumulation without a concomitant increase in feeding rate.

Figure 1. TGF-β Signaling Modulates Feeding Rate and Fat Storage in Adults

(A) TGF-β signaling pathway as deduced from genetic analyses of dauer formation. (B) Pharyngeal pumping rates of TGF-β pathway components under various conditions. Well-fed, pheromone untreated: daf-7(e1372) and daf-1(m40) pumped at 81% and 77% of wild-type rate, respectively. The reduced pumping rate of daf-1(m40) was suppressed by daf-3(mgDf90) but not by daf-12(m20). Food deprived, pheromone untreated: pumping rates of young adult wild type and all mutant genotypes were reduced to the same basal level. Well-fed, pheromone treated: treatment caused feeding reduction in well-fed wild type and daf-12 mutants. Similar treatment did not further reduce feeding rates of daf-7, daf-1, or daf-3 mutants. Standard deviation bars are shown. (C) Examples of Sudan black fat staining. Adult daf-1(m40) mutants had increased fat relative to wild type. This excess fat was suppressed by daf-3(mgDf90) but not by daf-12(m20). Excess fat of daf-1(m40) animals was reduced by expression of daf-1(+) in late larval/adult stage animals using an hsp16.2 heat-shock promoter. These images are representative of numerous animals from each genotype observed in multiple, independent experiments (see Methods). (D) Quantitation of relative amounts of Sudan black staining. Animals grown under identical conditions were fixed and stained using methods designed to minimize variation. For each genotype, average staining intensity of 10 animals is reported. Asterisks indicate statistical significance (p<0.001) as determined by t-test when comparing genotype of interest with daf-1(m40). Standard error bars are shown. (E) Rescue of reduced pumping rate by expression of daf-1 in late larval/adult stage daf-1(m40) mutants. Pumping rate changes were noted 0.5h and 24h after induction. Asterisks indicate statistically significant change (p<0.001 determined by ANOVA with Bonferroni post-test) relative to daf-1(m40). Standard deviation bars are shown

Figure 2. Reconstitution of daf-1 in RIM and RIC Interneurons Restores Wild-type Feeding Rate and Fat Storage to daf-1(m40) Mutants

Tissue-specific promoters listed in Table 1 were used to express full-length daf-1::gfp in various cell types of daf-1(m40) animals. (A) Examples of neuronal expression patterns of promoters used to target daf-1::gfp. Axis indicates P, posterior, A, anterior, D, dorsal, V, ventral. (B) Effects of tissue selective reconstitution of daf-1 on feeding rate. Asterisks indicate statistical significance relative to daf-1(m40) (p<0.001 as determined by ANOVA with Bonferroni post-test). Standard deviation bars are shown. (C) Effects of tissue selective reconstitution of daf-1 on fat. Quantitations for representative fat phenotypes shown here are reported in Figure S3. (D) P_daf-1::daf-1::gfp co-localized with P_tdc-1::RFP in RIM and RIC (white arrowheads).

Figure 3. Activation of daf-3 in RIM and RIC Causes Fat Accumulation despite Feeding Reduction

(A) Feeding rate. Asterisks indicate statistical significance (p<0.001 determined by ANOVA with Bonferroni post-test) relative to daf-1(m40); daf-3(mgDf90). Standard deviation bars are shown. (B–C) Fat content. Asterisks indicate statistical significance (p<0.001) as determined by t-test comparing genotype of interest with daf-1(m40); daf-3(mgDf90). Standard error bars are shown.

Figure 4. Tyramine and Octopamine Are Required for the Reduced Feeding Rate but not the Excess Fat of daf-1(m40) Mutants

(A) Effects of 4mM tyramine or octopamine on pumping rates of well-fed adult stage animals. Rescue of daf-1(m40) reduced feeding rate by each of tdc-1(ok914), tbh-1(ok1196), and ser-2(pk1357). Asterisks indicate statistical significance (p<0.001 as determined by ANOVA with Bonferroni post-test) for indicated comparisons. Standard deviation bars are shown. (B–C) tdc-1(ok914), tbh-1(ok1196), and daf-12(m20) mutations did not suppress the excess fat of daf-1(m40). Asterisks indicate statistical significance (p<0.001) as determined by t-test comparing genotype of interest and daf-1(m40). Standard error bars are shown. (D) Reconstitution of SER-2 tyraminergic receptor using tissue specific promoters in daf-(m40); ser-2(pk1357) mutants. Asterisks indicate statistical significance (p<0.001 determined by ANOVA with Bonferroni post-test) relative to non-transgenic daf-1(m40); ser-2(pk1357). Standard deviation bars are shown.

Figure 5. Mutations in GPCR Signaling and Metabotropic Glutamate Receptors Abrogate the Excess Fat but not Reduced Feeding of daf-7(

−). Increased fat but not reduced feeding rate of daf-7(e1372) was suppressed by mutations in goa-1, dgk-1, eat-4, mgl-1, and mgl-3 as well as a predicted gain of function mutation in egl-30. (A) Feeding rate. Asterisks indicate statistical significance relative to daf-7(e1372) (p<0.001 determined by ANOVA with Bonferroni post-test). Standard deviation bars are shown. (B) Representative examples of fat content in various mutants. (C) Fat quantitations. Asterisks indicate statistical significance (p<0.001) as determined by t-test when comparing genotype of interest with daf-7(e1372). Standard error bars are shown. (D) Expression sites of mgl-1 and mgl-3. P_mgl-1::mCherry was expressed in AIA amphid interneurons, RMDV and RMDD ring interneurons/motoneurons, and pharyngeal NSM serotonergic neurons. P_mgl-3::gfp was expressed in NSM, ADF, ASE, and AWC amphid sensory neurons, and the RIB and RIC interneurons. Occasional expression in BAG ciliated neurons was also noted.

Figure 6. Model for the TGF-β Neural Circuit of Fat and Feeding Regulation

The DAF-7 TGF-β ligand expression is only detected in the ASI pair of sensory neurons while its receptors, DAF-1 and DAF-4 are broadly expressed in the nervous system. During favorable environmental conditions DAF-7 is secreted from ASI and signaling to two pairs of synaptically distant interneurons, RIM and RIC, to inhibit the DAF-3 co-SMAD. This promotes wild-type growth, egg laying, food intake behavior, and fat accumulation. Adverse environmental conditions such as increased population density combined with reduced food availability inactivate daf-7, relieving inhibition of DAF-3. During early larval stage, this leads to dauer formation and requires the nuclear hormone receptor DAF-12. In adults, DAF-3 activation in RIM and RIC causes reduced feeding rate, egg retention, and increased fat. These processes are regulated through distinct signals. Subsequent to DAF-3 activation, tyramine, synthesized in RIM and RIC, and octopamine, synthesized in RIC, cause feeding reduction. Tyraminergic feeding reduction is mediated through activation of the SER-2 GPCR on a subset of pharyngeal neurons. Glutamate signaling through neuronally expressed metabotropic glutamate receptors, MGL-1 and MGL-3, mediates fat increasing effects of daf-7(−). Fat increase is ultimately associated with increased de novo fat synthesis in the periphery.