A branched-chain fatty acid is involved in post-embryonic growth control in parallel to the insulin receptor pathway and its biosynthesis is feedback-regulated in C. elegans

Abstract

Growth and development of multicellular organisms are controlled by signaling systems that sense nutrition availability and metabolic status. We report a novel and surprising factor in Caenorhabditis elegans development, the monomethyl branched-chain fatty acid C17ISO, a product of leucine catabolism. We show here that C17ISO is an essential constituent in a novel mechanism that acts in parallel with the food-sensing DAF-2 (insulin receptor)/DAF-16 (FOXO) signaling pathway to promote post-embryonic development, and that the two pathways converge on a common target repressing cell cycle. We show that C17ISO homeostasis is regulated by a SREBP-1c-mediated feedback mechanism that is different from the SREBP-1c-mediated regulation of common fatty acid biosynthesis, as well as by peptide uptake and transport. Our data suggest that C17ISO may act as a chemical/nutritional factor in a mechanism that regulates post-embryonic development in response to the metabolic state of the organism.

Figure 1.

C17ISO is required for post-embryonic development. (A) Progeny of the C17ISO-deficient elo-5(lf) adult uniformly entered L1 diapause (indicated by arrows). (B) The L1-arrested worms were rescued to full growth and propagation after adding C17ISO to the plates 2 d after the arrest. Arrowhead points to P0 animal surrounded by its progeny. (C) The C17ISO-depleted elo-5(lf) animals were maintained on OP50 plates supplemented with various concentrations of C17ISO from the time of hatching. (n) Number of animals counted in each experiment. Bars, 200 μm.

Figure 2.

C17ISO deficiency causes developmental arrest in early L1 independently from the DAF-2/DAF-16 food-sensing pathway. (A) The M-cell lineage during L1 development (data adapted from Sulston and Horvitz 1977). (B) hlh-8∷GFP expression in a single M cell in the C17ISO-deficient L1-arrested elo-5(RNAi) larva. (C) hlh-8∷GFP expression in the two divided M cells in a wild-type L1 growing larva. (D) The changes in the seam cell patterns during L1 (adopted with permission from Blackwell Publishing [© 2002; http://www.blackwell-synergy.com] from Knight et al. 2002). (E) AJM-1∷GFP expression pattern at adherens junctions in a C17ISO-deficient L1-arrested elo-5(RNAi) larva corresponds to a stage about 3 h post-hatching (Knight et al. 2002). (F) AJM-1∷GFP expression pattern in a wild-type growing L1 animal corresponding to about 12 h post-hatching. (G) The disconnected seam cell shapes, typical of animals ∼6 h post-hatching (D), were observed in starved daf-16(lf) mutants. (H) The seam cell shapes in daf-16(lf); elo-5(RNAi) are similar to that in elo-5(RNAi) (E), and correspond to a stage about 3.3 h post-hatching (D).

Figure 3.

C17ISO acts in parallel to the DAF-2/DAF-16 pathway to repress cki-1 expression in seam cells. (A–E) Images of L1 animals of various genotypes and culture conditions expressing an integrated cki-1∷GFP reporter in seam cells, as indicated. The animal in A is committed to growth, the animal in C was among progeny of animals that were exposed to elo-5(RNAi) for a lifetime, it is L1-arrested. The GFP expression in seam cells was prominent in L1 animals arrested by deficiency of mmBCFA or by starvation, but not in developing L1, irrelevant to the daf-16 function. Bars, 15 μM for all images.

Figure 4.

acs-1 expression is up-regulated by deficiency of C17ISO but not C13ISO, and this regulation requires transcription factors sbp-1 and cbp-1. (A–E) Images of worms expressing GFP from the acs-1Prom∷GFP reporter transgene. RNAi treatment and mmBCFA supplementations are indicated. A moderate expression of the reporter in a wild-type background (A) increased dramatically following the depletion of C17ISO [acs-1(RNAi) + C13ISO] (B). (C) The increase was abolished in the experiments where C13ISO supplementation was replaced with C17ISO. RNAi of sbp-1 (D), but not nhr-49 (E), inhibited the acs-1 up-regulation. Bars, 100 μM. (F) Results of a quantitative RT–PCR analysis of the endogenous acs-1 gene expression in C17ISO-deficient animals. The levels are relative to a wild-type sample. (G–L) Images showing the GFP expression from either the elo-5Prom∷GFP or the acs-1Prom∷GFP transgene in wild-type or RNAi-treated animals as indicated. RNAi of cbp-1inhibits the steady state elo-5 expression as well as the up-regulation of acs-1 by mmBCFA deficiency.

Figure 5.

pep-2(RNAi) and cbp-1(RNAi), but not mdt-15(RNAi), significantly influence mmBCFA biosynthesis. (A–D) GC analysis of FA content in total lipid extracts obtained from RNAi-treated strains as indicated. Arrowheads point to the C15ISO and C17ISO peaks. Drastic decreases in mmBCFA biosynthesis were observed in animals treated with pep-2(RNAi) and cbp-1(RNAi), but not animals with mdt-15(RNAi). By comparison, all three RNAi treatments affected the fractions of C20 PUFA. (E–J). GFP images showing the expression of the elo-5Promoter∷GFP or sbp-1Promoter∷GFP or acs1Promoter∷GFP transgene in the wild-type or pep-2(RNAi) strain as indicated. pep-2(RNAi) causes a significant reduction of the expression of elo-5∷GFP but not the expression of sbp-1∷GFP or asc1∷GFP. Bars, 100 μM.

Figure 6.

A model for a regulatory function and homeostasis of a long-chain mmBCFA in C. elegans. Arrows and T bars illustrate positive and negative interactions, respectively. The data provided in this study suggest that C. elegans has a novel regulatory mechanism that involves mmBCFA to control the onset of post-embryonic growth and development. C17ISO is required to repress cki-1 and other growth regulators through the interaction with an unknown factor(s) (X). This mechanism functions in parallel to the well-known insulin receptor pathway that is capable of sensing food (Gems et al. 1998). The diagram also indicates a feedback regulatory circuit that plays a critical role in C17ISO homeostasis. C17ISO may represent critical metabolic and growth conditions that include the levels of mmBCFA and essential amino acids, protein uptake and transport, and certain lipid metabolic conditions.