Hexokinase regulates Mondo-mediated longevity via the PPP and organellar dynamics

Reviewer #1 (Public Review):

In this manuscript, Laboy and colleagues investigated upstream regulators of MML-1/Mondo, a key transcription factor that regulates aging and metabolism, using the nematode C. elegans and cultured mammalian cells. By performing a targeted RNAi screen for genes encoding enzymes in glucose metabolism, the authors found that two hexokinases, HXK-1 and HXK-2, regulate nuclear localization of MML-1 in C. elegans. The authors showed that knockdown of hxk-1 and hxk-2 suppressed longevity caused by germline-deficient glp-1 mutations. The authors demonstrated that genetic or pharmacological inhibition of hexokinases decreased nuclear localization of MML-1, via promoting mitochondrial β-oxidation of fatty acids. They found that genetic inhibition of hxk-2 changed the localization of MML-1 from the nucleus to mitochondria and lipid droplets by activating pentose phosphate pathway (PPP). The authors further showed that the inhibition of PPP increased the nuclear localization of mammalian MondoA in cultured human cells under starvation conditions, suggesting the underlying mechanism is evolutionarily conserved. This paper provides compelling evidence for the mechanisms by which novel upstream metabolic pathways regulate MML-1/Mondo, a key transcription factor for longevity and glucose homeostasis, through altering organelle communications, using two different experimental systems, C. elegans and mammalian cells. This paper will be of interest to a broad range of biologists who work on aging, metabolism, and transcriptional regulation.

Reviewer #2 (Public Review):

Raymond Laboy et.al explored how transcriptional Mondo/Max-like complex (MML-1/MXL-2) is regulated by glucose metabolic signals using germ-line removal longevity model. They believed that MML-1/MXL-2 integrated multiple longevity pathways through nutrient sensing and therefore screened the glucose metabolic enzymes that regulated MML-1 nuclear localization. Hexokinase 1 and 2 were identified as the most vigorous regulators, which function through mitochondrial beta-oxidation and the pentose phosphate pathway (PPP), respectively. MML-1 localized to mitochondria associated with lipid droplets (LD), and MML-1 nuclear localization was correlated with LD size and metabolism. Their findings are interesting and may help us to further explore the mechanisms in multiple longevity models. The data support their proposed working model. Nonetheless, the roles of hxk-1 and lipid oxidation in regulating LD, as proposed in the working model, are not clear.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

In this manuscript entitled "Hexokinase regulates Mondo-mediated longevity via the PPP and organellar dynamics", Laboy and colleagues investigated upstream regulators of MML-1/Mondo, a key transcription factor that regulates aging and metabolism, using the nematode C. elegans and cultured mammalian cells. By performing a targeted RNAi screen for genes encoding enzymes in glucose metabolism, the authors found that two hexokinases, HXK-1 and HXK-2, regulate nuclear localization of MML-1 in C. elegans. The authors showed that knockdown of hxk-1 and hxk-2 suppressed longevity caused by germline-deficient glp-1 mutations. The authors demonstrated that genetic or pharmacological inhibition of hexokinases decreased nuclear localization of MML-1, via promoting mitochondrial β-oxidation of fatty acids. They found that genetic inhibition of hxk-2 changed the localization of MML-1 from the nucleus to mitochondria and lipid droplets by activating pentose phosphate pathway (PPP). The authors further showed that the inhibition of PPP increased the nuclear localization of mammalian MondoA in cultured human cells under starvation conditions, suggesting the underlying mechanism is evolutionarily conserved. This paper provides compelling evidence for the mechanisms by which novel upstream metabolic pathways regulate MML-1/Mondo, a key transcription factor for longevity and glucose homeostasis, through altering organelle communications, using two different experimental systems, C. elegans and mammalian cells. This paper will be of interest to a broad range of biologists who work on aging, metabolism, and transcriptional regulation.

Reviewer #2 (Public Review):

Raymond Laboy et.al explored how transcriptional Mondo/Max-like complex (MML-1/MXL-2) is regulated by glucose metabolic signals using germ-line removal longevity model. They believed that MML-1/MXL-2 integrated multiple longevity pathways through nutrient sensing and therefore screened the glucose metabolic enzymes that regulated MML-1 nuclear localization. Hexokinase 1 and 2 were identified as the most vigorous regulators, which function through mitochondrial beta-oxidation and the pentose phosphate pathway (PPP), respectively. MML-1 localized to mitochondria associated with lipid droplets (LD), and MML-1 nuclear localization was correlated with LD size and metabolism. Their findings are interesting and may help us to further explore the mechanisms in multiple longevity models, however, the study is not complete and the working model remains obscure. For example, the exact metabolites that account for the direct regulation of MML-1 were not identified, and more detailed studies of the related cellular processes are needed.

The identification of responsible metabolites is necessary since multiple pieces of evidence from the study suggests that lipid other than glucose metabolites may be more likely to be the direct regulator of MML-1 and HXK regulate MML-1 indirectly by affecting the lipid metabolism: 1) inhibiting the PPP is sufficient to rescue MML-1 function independent of G6P levels; 2) HXK-1 regulates MML-1 by increasing fatty acid beta-oxidation; 3) LD size correlates with MML-1 nuclear localization and LD metabolism can directly regulate MML-1. The identification of metabolites will be helpful for understanding the mechanism.

Beta-oxidation and the PPP are involved in the regulation of MML-1 by HXK-1 and HXK-2, respectively. But how these two pathways participate in the regulation is not clear. Is it the beta-oxidation rate or the intermediate metabolites that matters? As for the PPP, it provides substrates for nucleotide synthesis and also its product NADPH is essential for redox balance. Is one of the metabolites or the NADPH levels involved in MML-1 regulation? More studies are needed to provide answers to these concerns.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Following are my comments that the authors may want to address to further improve this excellent paper.

Major comments

(1) Although the authors provided evidence that hexokinases in glucose metabolism are associated with germline-deficient glp-1(-) mutants, they did not mention why they focused on glp-1(-) mutants rather than other longevity mutants. In their previous study (Nakamura et al., 2016), they showed that MML-1 is required for multiple longevity pathways in C. elegans, including reduced mitochondrial respiration and insulin/IGF-1 signaling. Please discuss why the authors focused on glp-1(-) mutants in this paper. It will be even better if the authors test the roles of hexokinases in some other longevity regimens.

Many thanks for this astute comment. Previously we had shown that mml-1 is required for glp-1, daf-2, and isp-1 longevity, and Johnson et al. had shown a requirement for eat-2, hence the idea that MML-1 is a convergent transcription factor. We first focused on glp-1 because that was the starting point of our screen, and the result was clear and simple: hexokinases regulate MML‑1 nuclear localization and activity in glp-1 and are required for longevity. Naturally, the question arises: do hexokinases behave like MML-1 as convergent longevity regulators across pathways? To address this, we examined the interaction of hxk-1 and hxk-2 with isp-1, daf-2, and raga-1. Specifically, we now show that:

A. Like glp-1(e2141) mutants, isp-1(qm150) mutants stimulate MML-1 nuclear localization, and the hexokinases are required for isp-1 longevity (Figure 1G-H).

B. daf-2(e1370) mutants do not further stimulate MML-1 nuclear localization beyond basal levels, yet MML-1 is strongly required for daf-2 longevity (Nakamura et al., 2016, Supplementary Figure 1L-M). However, the hexokinases are not required for daf-2 longevity (Supplementary Figure 1M), suggesting that the signaling pathway is wired differently in daf-2, and that other pathways regulate MML-1 activity.

C. raga-1(ok701) mutants stimulate MML-1 nuclear localization and mml-1 is required for raga-1 longevity, suggesting that MML-1 acts downstream of TORC1 signaling (Supplementary Figure 1N-O). However, hexokinases are not required for raga-1 longevity, suggesting that raga-1 acts downstream or parallel to hexokinase signaling (Supplementary Figure 1P).

D. We performed untargeted metabolomics in glp-1, daf-2, and mml-1 single and double mutants and observed that hexose phosphates, which have been shown to regulate MML-1 human homologs MondoA/ChREBP, were differentially regulated between mutants.

Author response image 1.

E. Altogether these experiments reveal that though MML-1 promotes longevity in most pathways, the hexokinases are only required in some (glp-1, isp-1), but not others (raga-1, daf-2). Furthermore, strong MML-1 nuclear localization is often but not always associated with longevity (e.g. daf-2), and the wiring of the signaling pathway is different for various longevity regimens. Consistently, mTOR and Insulin signaling are more functionally linked and therefore may show a more similar genetic profile. Differences in hexose phosphate between glp-1 and daf-2 could explain why MML-1 requires hexokinase function in glp-1 to promote longevity but not in daf-2. However, considerably more work is required to rigorously validate this hypothesis.

(2) In figure 5, the authors investigated whether the association between PPP and MML‑1/MondoA, tested in C. elegans, is conserved in mammals under starvation conditions. The authors should clarify why they tested the MondoA localization upon starvation in cultured human cells. This comment is related to my comment #1 as the authors could determine the roles of hexokinases under dietary restriction (DR)-conditions or in DR-mimetic in eat-2(-) mutants.

In this case, the actual translatability to a worm longevity pathway was not our goal. Rather, we examined MondoA in cell culture under contrasting conditions of MondoA subcellular localization, where high glucose media had cytosolic/nuclear localization and starvation conditions cytosolic localization. We then showed that similar to our data in worms, PPP inhibition with 6-AN induced MondoA nuclear localization and activity. We now mention this rationale in the results section, lines 352-356.

(3) In figure 2, the authors showed that HXK-2 regulates mitochondrial localization of MML-1, and HXK-1 regulates nuclear localization of MML-1 through mitochondrial β-oxidation in glp‑1(-) mutants. Can the authors test whether mitochondrial β-oxidation affects the effects of hxk RNAi on longevity of glp-1(-) mutants?

Excellent suggestion. We tried to test this idea and found that acs-2 RNAi alone abolished glp-1 longevity, making epistasis experiments difficult to interpret. This is consistent with published data showing that glp-1 longevity requires NHR-49, a transcription factor that regulates mitochondrial b‑oxidation, that drives acs-2 expression (Ratnappan et al., 2014). It could well be that b‑oxidation inhibition promotes MML-1 nuclear localization but abolishes lifespan extension because of epistatic effects on other transcription factors or processes. Further investigation would be required to elucidate the exact mechanism that goes beyond the scope of the paper.

(4) The authors showed that 2-deoxy-glucose, which decreases the activity of HXK, decreased the nuclear localization of MML-1, and this is consistent with their genetic data. Based on these data, 2-deoxy-glucose is expected to decrease longevity. Interestingly, however, 2-deoxy-glucose has been reported to increase lifespan by restricting glucose, whereas extra glucose intake decreases lifespan in C. elegans, shown by multiple research groups, including M. Ristow, C. Kenyon, and S.J.V. Lee labs. This is seemingly paradoxical and worth discussing with key references, especially because MondoA and Chrebp are known as glucose-responsive transcription factors.

Thank you for this important comment. 2-DG has been shown to extend lifespan by suppressing glucose metabolism at concentrations ranging from 0.1 to 5 mM, higher concentrations ranging from 20 to 50 mM had the opposite effect decreasing lifespan (Schulz et al., 2007). The concentration we tested was 50 mM 2-DG and observed decreased MML-1 nuclear localization, which is consistent with the previous data showing decreased longevity. We now raise this point in the discussion suggesting that mild inhibition of glucose metabolism has beneficial effects on longevity, while strong suppression causes a shortening of the lifespan (lines 411-414).

Minor comments

(1) The current Introduction does not include the explicit statement about that MML-1 and MondoA are homologs. Please clarify this as naive readers may be confused.

Thank you for pointing this out. We now say in the intro that MondoA and MML-1 are homologs (lines 59-60).

(2) In figure 1, the effects of hxk-3 on nuclear localization of MML-1 is small compared to those of hxk-1 and hxk-2. Please add speculation about why HXK-3 has different roles in nuclear localization of MML-1 compared to HXK-1 and HXK-2.

According to GExplore 1.4 (Hutter & Suh, 2016), hxk-3 expression declines during larval development and is low expressed in the adult. Perhaps it has little effect in the young adult, and the other hexokinases suffice to support MML-1 nuclear localization. It also remains possible that hxk-3 is not required in glp-1, but required in other longevity pathways.

(3) The authors tested the effects of genetic inhibition of hxk-1 and hxk-2 on the regulation of MML-1 localization and lifespan of glp-1(-) mutants by using RNAi. I wonder whether the authors can perform the experiments with hxk-1 or hxk-2 loss (or reduction) of function mutants. If they cannot, please discuss the reason and the limitations of RNAi.

This is an important point raised by the reviewer. We found that RNAi was most effective for phenotypes related to MML-1 nuclear localization and longevity, likely because it results in acute knockdown. We also showed that pharmacological inhibition of hexokinase function with 3BrP and 2‑DG (Supplementary Figure 1B and 1C) and the PPP with 6-AN (Figure 3B) had consistent results with our observation with RNAi.

We generated hexokinase KO mutants by deleting the coding sequence of each hexokinase by CRISPR/Cas9. First, we measured the expression of each hexokinase isozyme in each mutant. Notably, hxk-1(syb1271) null mutant had higher expression of hxk-2 and hxk-3, hxk-2(syb1261) did not significantly affect the expression of hxk-1 and hxk-3, and hxk-3(syb1267) had a mild increase in hxk-2 expression. We followed up on the hxk-1(syb1271) and hxk-2(syb1261) and crossed these mutants with our MML-1::GFP reporter. We observed a modest but significant reduction in MML-1 nuclear localization in both strains. The effect with RNAi is much stronger in comparison to the null mutants, potentially due to a compensatory upregulation of the other hexokinases in the mutants that we do not observe with RNAi (Supplementary Figure 1D-E). Another alternative is that there is a threshold in the effects of hexokinase function on MML-1 nuclear localization. We tried to generate a hxk-1; hxk-2 double mutant but it was lethal and therefore did not pursue this further.

Author response image 2.

(4) Please correct minor typos throughout the manuscript. Following are some examples.
- On page 4, line 111, please correct "Supplementary Figure D-E" to "Supplementary Figure 1D-E".

- On page 9, line 272, please correct "3A-B" to "4A-B".

- On page 9, line 275, please correct "S4" to "4".

- On page 10, line 309, please correct "4A" to "4B"

Corrected.

(5) In Fig. 3E, please add the information about the scale bars in figure legends.

Corrected.

Reviewer #2 (Recommendations For The Authors):

Here are some detailed suggestions for the authors:

(1) Since MML-1/MXL-2 complex functions in multiple longevity models, e.g. DR, ILS, what are the roles of HXK-1 and HXK-2 in these models?

We now show that although mml-1 is required in most longevity pathways, hxk-1 and hxk-2 are required in some pathways (glp-1, isp-1) but not others (daf-2, raga-1). See above for more details.

(2) As for the metabolites screening, the lipid metabolic genes can be included. Not only for the above reasons, also previous study had found that the mml-1 mRNA levels and MML-1 GFP nuclear localization were all increased in the glp-1 model, while mml-1 mRNA levels were unaffected by hxk knockdown, suggesting more pathways be involved.

We agree with the reviewer that understanding what metabolites regulate MML-1 nuclear localization and activity is an important, yet challenging question. Our studies demonstrate a role of glucose metabolism, in particular, hexokinase in this process, consistent with hexose-p being activators of MondoA. Our data also suggest mechanisms beyond hexose-p regulate MML-1, since knockdown of the PPP components stimulates MML-1 even when hxk-2 is depleted and low G6P, and inhibition of the PPP with 6-AN stimulates MondoA nuclear localization under starvation conditions in mammalian cell culture. We tested redox regulation, nucleoside, and lipid metabolism as candidate processes (see below). Notably, our data suggest this other mechanism is tied to lipid metabolism through droplet size since various perturbations that impact LD size and number (atgl-1, dgat-2, tkt-1, Figure 4) affected MML-1 nuclear localization. It remains an open question whether MML-1 is regulated by other metabolites through a ligand-protein interaction or not. We cannot exclude that beyond lipid droplet regulation, specific lipids, other metabolites, or metabolic modules linked to the PPP might regulate MML-1 nuclear localization and activity.

We employed genetic manipulation and pharmacological inhibition to understand the upstream signals that regulate MML-1. These approaches will not be sufficient to determine whether other metabolite(s) are involved in MML-1/MondoA translocation to the nucleus through a direct interaction. Novel technologies that determine protein-metabolite interactions (e.g. MIDAS) will help us answer this question in future work, and go beyond the scope of this paper. As a compromise, we discuss possible metabolites that may orchestrate this based on our observations based on MML‑1 subcellular localization at LD/mitochondria (including PPP and TCA cycle intermediates).

(3) Line 238, it should be "NADPH".

Corrected.

(4) RNAi targeting enzymes of different branches of PPP can be performed

In our initial screen, we examined the effect of various enzymes of the PPP on MML-1 nuclear localization (Figure 1A, Supplementary Table S1) and found that knockdown of enzymes in both the oxidative phase (PGDH/T25B9.9) and non-oxidative phase (transketolase/TKT-1) affect MML-1 nuclear localization. In line, 6-AN treatment, which affects the oxidative phase, also stimulated MML‑1 nuclear localization (Figure 3B). We also observed that knockdown of enzymes involved in ribose 5P conversion to ribose, ribose 1P, and phosphoribosyl pyrophosphate, an intermediate in nucleotide biosynthesis, decreased MML-1 nuclear localization (rpia-1, _F07A11._5, Y43F4B.5, _R151._2; Supplementary Table S1). Whether MML‑1/MondoA responds to nucleotide pool remains elusive.

(5) As for PPP, these are many possibilities that can be tested. For example, as PPP supplies NADPH for oxidative balance, does MML-1 respond to ROS? Also, it appears the genes in the non-oxidative arm of PPP regulate MML-1, so is nucleotide synthesis involved?

Thank you for the suggestion. We tested other enzymes involved in NADPH production from the folate cycle and observed a mild but significant reduction of MML-1 nuclear localization upon dao-3i (Supplementary Table S1). Moreover, we tested whether MML-1 nuclear localization is responsive to ROS. While paraquat exposure induced oxidative stress by measuring the transcriptional reporter gst‑4p::GFP (Supplementary Figure 3A), paraquat exposure did not significantly affect MML-1 nuclear localization (Supplementary Figure 3B). Therefore we think it less likely that NADPH production acting through redox regulation is the main effect.

We also tried supplementation with some of the metabolite outputs of PPP including ribose, ribulose, and xylulose, as well as nucleosides (see below), but saw no effect on MML-1 nuclear localization. We agree that further studies are required to pinpoint whether there is another metabolic moiety regulating MML-1 at the protein-ligand level, but this goes beyond the scope of the current investigation.

Author response image 2.