Dietary bacteria control C. elegans fat content through pathways converging at phosphatidylcholine

Reviewer #1 (Public Review):

Summary:

This paper reports the finding that less fat accumulates in C. elegans that are feeding on Comamonas aquatica DA1877 (DA) vs the standard lab diet of Escherichia coli OP50 (OP50). While these bacteria are likely to be different in many ways, the authors found that fat accumulation phenotype depends on the vitamin B12 content of the bacterial diet and the involvement of B12 in the methionine cycle, affecting SAMS-1 and phosphatidylcholine (PC) synthesis. They report that low PC levels activate SREBP-1 (SBP-1 in C. elegans) and that an important target of SBP-1 is the delta 9 desaturase FAT-7. Finally, they describe a role for ASM-3, an acid sphingomyelinase, in influencing PC synthesis and fat accumulation in the worm.

Strengths:

This is a comprehensive story about how a dietary change affects fat accumulation in C. elegans. Their experimental evidence is convincing. The most novel aspect of this paper is that the coelomecyte expression of asm-3 contributes to PC/TAG homeostasis in C. elegans, which most likely occurs through the production of phosphocholine by the enzymatic breakdown of sphingomyelin by ASM-3. The phosphocholine will provide precursors for phosphatidylcholine (PC) synthesis, contributing to the PC synthesis pathway.

Weaknesses:

In the way the story is presented, the authors tend to imply that they discovered the pathways of B12, PC, SBP-1, and FAT-7, ignoring some important studies describing the relationship between PC synthesis and TAG accumulation in both the mammalian lipid metabolism field (liver) as well as in C. elegans. Many previous studies with similar results are not cited appropriately. Thus, the pathways reported in the paper are not new, and in this sense, the work is mostly confirmatory.

Reviewer #2 (Public Review):

Summary:

Han et al. present a manuscript focusing on difference metabolism and the regulatory circuits controlling it in C. elegans fed two bacterial diets. In the first three figures and a half figures, using a combination of methods, they investigate lipid levels, changes in gene expression and genetic assays to come to the conclusion that vitamin B12 acts through the S-adenosylmethioine synthase sams-1 to perturb phosphatidylcholine levels, which in turn stimulate the C. elegans ortholog of the SREBP transcription factors to activate fatty acid synthesis genes such as fat-7/SCD1. Thus, while connections between diet, metabolic pathways and gene regulation is of general interest, this study largely confirms the work of others without direct credit in many instances, then fails to develop a more novel cell non-autonomous link between the pathways in the last two figures. Thus, this study would be expected to have a useful impact on the field, if it can be placed in context of previously published work.

Strengths:

(1) Connections between diet, metabolic pathways and gene regulation is of general interest
(2) Figures 1-4 confirm data/observations from previously published work from MacNeil, et al. Cell 2015; Walker, et al. Cell 2011; Svensk, et al. PLoS Genetics 2013; Smulan, et al. Cell Reports, 2016; Giese, et al. eLife 2020 and Qin, et al. Cell Reports 2022..
(3) The data in figures 5 and 6 showing importance of non-cell autonomous effects on metabolism.

Weaknesses:

(1) In order to differentiate their study from previous work, it seems that the authors try to make the argument that PC is higher in Comomonas than E. coli, therefore they are looking at repression of SBP-1-dependent function, however, the pairing of the diets is arbitrary, and the comparisons could easily be reversed. They are simply comparing a higher to a lower level of PC, rather than a basal to a lower, thus the concepts are the same. In addition, they fail to cite the larger body of literature linking phospholipid balance to SREBP function. For example, multiple studies in mammalian models link phospholipid balance, not just lowered PC, to SREBP function: Lim, Genes and Dev 2011; Wang, et al. Cell Stem Cell, 2018; Rong, et al. J Clin Invest 2017; Smulan et al, Cell Reports, 2016; Dobrosotskaya, Science. 2002 and recently, Rong, et al. Cell Met 2024.

(2) Figure 1: For example, the data in figure 1, shows measures of lipid content, RNA seq showing changes in metabolic enzymes such as fat-7/SCD-1 and lipid levels have already been shown in MacNeil, et al. Cell 2013 (lipid levels and gene expression changes) and the lipid levels in Comomonas vs E. coli were published in Ditot, et al. Nature Communications 2022 by Dr. Marian Walhout's lab.

(3) Figure 2/3: In Figure 2 and 3, they use a genetic screen to find regulators of fat-7/scd1 expression, and unsurprisingly, pull out genes with known to regulate this pathway. The authors go on to show that changes in SAM lead to changes in PC, and affect SBP-1/SREBP-1-dependent lipogenesis. This is a well described pathway from publications by the Walhout lab, Dr. Amy Walker's lab and Dr. Marc Pilon's lab (Walker, et al. Cell 2011; Svensk, et al. PLoS Genetics 2013; Smulan, et al. Cell Reports, 2016; Giese, et al. eLife 2020) in addition to a recent publication, Qin, et al. Cell Reports 2022. While some of these studies are cited in other places in the manuscript, the authors describe their results as "discovery", then fail to cite the relevant studies at those points (selected examples below

(4) Selected examples of citation issues:

a) Selected example: pg 6: "To understand the mechanism underlying the regulation of host lipid content triggered by DA, we examined the gene expression changes elicited by the two different bacterial diets in young adult animals by RNA-seq...In particular, genes related to the biosynthesis of unsaturated fatty acids showed a significant decrease in expression in DA-fed worms. For example, the delta-(9) fatty acid desaturases, fat-5 and fat-7, (which convert fatty acids 16:0 to 16:1n7 and 18:0 to 18:1n9, respectively32) decreased"

MacNeil et al Cell 2013 published a transcriptomics comparing young adult DA and Op50, which demonstrated decreases in fat-5 and fat-7. While MacNeil is cited in other parts of the paper, since the authors have performed a highly similar experiment and obtained similar results, this should be described as confirming the MacNeil study rather than as new data.

b) Selected Example: pg 10: "To determine whether PC levels have a causal effect on organismal lipid content, we supplemented worm diets with choline, the PC precursor, and uncovered a dose-dependent decrease in lipid content as measured by O.R.O staining (Figure 3B)."

Addition of choline to supplement defects in PC synthesis was first shown by Brendza, et al. Biochem J 2007. It was confirmed in Walker, et al. 2011, and further confirmation of PC rescue show in Ding, et al. 2015. The Brendza study is not cited at all and while studies from the Walker lab are cited in other places, the authors omit that changes in the DA diet are the same as changes seen when choline rescues PC loss from other perturbations.

c) Selected Example: pg 9: "Notably, DA has been reported as a B12-rich bacterium compared to OP16, hinting at the possibility that the DA diet might boost dietary B12 levels."

Reference 16 is Watson, et al. Cell 2015 where the Walhout lab demonstrates that DA does in fact act through the diet to alter the Met/SAM cycle and other B12 dependent processes in C. elegans. This paper, along with MacNeil above broke ground in linking B12 and the Met/SAM cycle to specific phenotypes in C. elegans, which was followed up by extensive work from the Walhout lab on this cycle, thus, it seems odd that the authors describe their own data as "hinting" at this connection.

d) Selected example: pg 17: "Indeed, this is further supported by our observation that mutants of histone methyltransferases SET-2 and SET-30 (which install H3K4me1 and H3K4me2, respectively) exhibited elevated lipid content on DA diet (data not shown). Notably, while both set-2 and set-30 mutants had this effect, only set-2 appears to control fat-7 expression (data not shown)". Extensive work from Dr. Anne Brunet's lab (Greer, et al. Nature 2010; Greer, et al. Nature 2011; Han, et al. Nature 2017) link set-2 and H3K4 methylation to lipid accumulation and fat-7. The authors fail to cite these studies.

Reviewer #3 (Public Review):

Summary:

The authors presented data that linked vitamin B12, S-adenosyl methionine (SAM), and phosphatidylcholine (PC) synthesis to lipid homeostasis in C. elegans. They confirmed mechanisms previously shown by other labs, including the regulation of FAT-7 expression by SBP-1, and the targeting of SEIP-1 by PC levels. The authors also attempted to link the synthesis of phospho-choline by the ASM-3 sphingomyelinase to PC synthesis and lipid homeostasis. However, the relative contribution of phospho-choline by ASM-3 versus the canonical Kennedy pathway was not elucidated. Therefore, the significance of the ASM-3-dependent mechanism to PC synthesis requires further investigation.

Strengths:

The authors used a wide range of biochemical and cell biological methods to measure fatty acid composition, neutral lipid levels, and lipid droplet dynamics in C. elegans. The quality of the data is generally high.

Weaknesses:

Data interpretation and the construction of the working model did not seem to take into account the two well-established pathways for PC synthesis. The Kennedy pathway generates PC from phospho-choline and DAG via a cytidine-based intermediate. The second PC synthesis pathway entails the methylation of PE by PEMT, with the donor methyl groups provided by the vitamin B12-dependent 1-carbon cycle. The authors' model seemed to overlook part of the Kennedy pathway that involves choline kinase (and not ASM-3) as the canonical enzyme that generates phospho-choline. The authors also did not explicitly consider DAG as a precursor of triacylglycerol (TAG), which was directly or indirectly measured as a readout of organismal fat content in the paper. Therefore, alternative models should be entertained. For example, the proposed genetic and dietary effects on lipid homeostasis could stem from the competition for a limiting pool of precursors that were shared by PC and TAG synthesis. PC itself may not have a deterministic role, as depicted by the authors' model. Finally, the claim that "coelomocytes regulate diets-induced lipid homeostasis through asm-3" was not well supported. In the absence of quantitative analysis of phospho-choline in mutants, it was unclear how much ASM-3 contributed to the overall phospho-choline, and ultimately PC level. The proposed inter-tissue regulation of PC synthesis also requires coelomocytes-specific knock-down/depletion of asm-3 for verification.