Hexokinases are required for germline longevity through the regulation of MML-1 localization and function.

A. Screening for MML-1 regulators upon knockdown of glucose metabolism enzymes. For clarity, we only mapped the candidate genes involved in glycolysis, TCA cycle, and PPP. See Supplementary Table S1 for a complete list of genes tested. MML-1::GFP (dhIs989) nuclear localization was measured in intestinal cells of day one adult worms grown in the RNAi egg-on. Each circle represents one gene encoding an isozyme or subunit of the complex. Circles are color-coded to indicate the increase (red) or decrease (blue) of MML-1::GFP nuclear localization upon knockdown of the enzymes compared to internal luci controls. DHAP, dihydroxyacetone phosphate; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle; PPP, pentose phosphate pathway. B. Quantification of MML-1 nuclear localization upon knockdown of glucose metabolism genes. Only genes that statistically affected MML-1 nuclear localization compared to the internal luci control are depicted (p<0.05; N≥2). C-D. Representative images (C) and quantification (D) of MML-1 nuclear localization in wildtype and the germline-less glp-1(e2141) longevity background upon knockdown of luci (control), hxk-1i, hxk-2i, and mml-1i. MML-1::GFP was quantified in nuclei from intestinal cells (white arrows) in day one adult worms (N=3). Scale bars, 10 μm. E. Relative mRNA levels of MML-1 downstream target mdl-1 in young adult worms measured by qPCR (N=3). F. Lifespans of wildtype and glp-1(e2141) upon hxk-1 and hxk-2 knockdown. Lifespans for hxk-1 and hxk-2 knockdown were performed in the same experiment and plotted separately for clarity; therefore, control lifespans are shared between plots. G. Quantification of MML-1 nuclear localization in wildtype and isp-1(qm150) mutants upon luci, hxk-1i, and hxk-2i in day one adult worms (N=3). H. Lifespan of wildtype and isp-1(qm150) upon hxk-1 and hxk-2 knockdown (N=2). Statistical significance was calculated with t-test in A and B, one-way ANOVA in D and G, two-tailed t-test in E, and Log-rank (Mantel-Cox) test in F and H. D and G bars represent mean ± SD, and E bars represent mean ± SEM.

Enhanced mitochondrial β-oxidation suppresses MML-1 nuclear localization upon hxk-1 knockdown.

A-B. Tissue expression of HXK-1::mKate2 (A) and HXK-2::mKate2 (B) isozymes. The primary tissues are identified with arrows. Scale bars, 20 μm. C. Colocalization of mitochondrial reporter myo-3p::mitoGFP with full-length HXK-2::mKate2 and d10 HXK-2::mKate2 lacking the first ten residues of the N-terminus. Scale bars, 10 μm. D. Representative confocal images of day one adult worms expressing MML-1::GFP and the mitochondrial reporter ges-1p::TOMM-20::mCherry under luci and hxk-2i. Scale bars, 10 μm. E. Intensity plot of the region of interest (ROI) from hxk-2i (D). F. Colocalization of MML-1::GFP and TOMM-20::mCherry in luci and hxk-2i. The colocalization analysis was calculated with the thresholded Mander’s colocalization coefficients tM1 (GFP channel) and tM2 (mCherry channel) (N=3). G. When glycolysis is reduced, cells rely on fatty acids for energy production. Free fatty acids are activated to acyl-CoA and transported to the mitochondria through the CPT in the mitochondrial outer membrane. The acyl-CoA is then metabolized to generate acetyl-CoA that can enter the TCA cycle to produce energy. MPC, mitochondrial pyruvate carrier; CPT, carnitine palmitoyltransferase. H-I. Oxygen consumption rate (OCR) under basal conditions (H) and after the addition of the mitochondrial β-oxidation inhibitor etomoxir (I). Mitochondrial β-oxidation is calculated as the percentage of decreased OCR after the injection of etomoxir (N=3). J. MML-1 nuclear localization of day one adult worms grown with double knockdown of hxk-1 or hxk-2 combined acs-2i (N=3). K. MML-1 nuclear localization of day one adult worms grown upon hxk-1 and hxk-2 knockdown with supplementation of etomoxir 100 μM egg-on (N=3). Statistical significance was calculated with t-test in F, and one-way ANOVA in H-K. F and J-K bars represent mean ± SD, H-I bars represent mean ± SEM.

HXK-2 regulates MML-1 localization and function through inhibition of the PPP.

A. The PPP is a cytosolic pathway that metabolizes G6P to produce NADPH and ribose-5P, and interconverts 3 to 7 carbon sugars. 6-Aminonicotinamide (6-AN) is a PPP inhibitor that acts on the NADPH-producing enzymes GSPD-1 and T25B9.9. B. Quantification of MML-1 nuclear localization of day one adult worms supplemented with 500 μM and 1 mM of 6-AN egg-on (N=3). C. Quantification of NADP+ and NADPH levels in day one adult glp-1(e2141) worms grown under hexokinase knockdown. T25B9.9i was used as a control. Metabolite levels were normalized to total protein concentration (N=3). D. Transketolase enzymatic activity assay in day one adult glp-1(e2141) worms grown under hexokinase knockdown. tkt-1i was used as a control. Activity levels were normalized to total protein concentration (N=3). E-F. Representative images (E) and quantification (F) of MML-1 nuclear localization of day one adult worms upon double knockdown of hxk-2 with the PPP enzymes T25B9.9 and tkt-1 (N=3). Scale bars, 20 μm. G. Relative mRNA levels of MML-1 downstream target mdl-1 in young adult worms upon double knockdown of hxk-2 in combination with enzymes of the PPP measured by qPCR (N=3). H. Quantification of G6P levels relative to total protein measured in day one adult glp-1(e2141) upon double knockdown of hxk-2 in combination with enzymes of the PPP (N=4). I. Lifespan analysis of glp-1(e2141) with hxk-2i and tkt-1i single and double knockdowns. Lifespan was performed in the same experiment as Supplementary Figure 3J; therefore, control lifespans are shared between plots (N=3). Statistical significance was calculated with a one-way ANOVA test in B-D and F-H, and Log-rank (Mantel-Cox) test in I. B and F bars represent mean ± SD, C-D and G-H bars represent mean ± SEM.

hxk-2 knockdown increases MML-1 colocalization with LD.

A. Anti-FLAG immunoprecipitation (IP) from whole-worm lysates of animals expressing MML-1 endogenously tagged with 3xFLAG in wildtype background. IP was analyzed by mass spectrometry (LC-MS/MS). The 25 top candidates co-enriched with MML-1 compared to non-transgenic wildtype control are shown as log FC, including MML-1 (blue) and its established binding partner MXL-2 (red). B. Crosstalk between glycolysis, PPP, and lipid droplet (LD) metabolism. On the one hand, LD expansion is favored by increased fatty acid synthesis and subsequent formation of triacylglycerol (TAG) by the diacylglycerol acetyltransferase DGAT-2. On the other hand, LD consumption releases free fatty acids from TAG by the rate-limiting lipase ATGL-1. C. Confocal microscopy of day one adult worms expressing MML-1::GFP and the mitochondrial reporter TOMM-20::mCherry stained with the LD dye LipiBlue under luci and hxk-2i (N=3). Scale bars, 10 μm. D. Intensity plot of the region of interest (ROI) from hxk-2i (C). E. Colocalization of MML-1::GFP and LD in luci and hxk-2i. The colocalization analysis was calculated with the thresholded Mander’s colocalization coefficients tM1 (GFP channel) and tM2 (LipiBlue channel) (N=3). F. Quantification of LD diameter in day one adult worms expressing the LD reporter PLIN-1::mCherry upon knockdown of hxk-2 and tkt-1 (N=3). G. Quantification of MML-1 nuclear localization under knockdown of the LD metabolic enzymes dgat-2 and atg-1 (N=3). H. Quantification of MML-1 nuclear localization upon double knockdown of tkt-1 and atgl-1 (N=3). Statistical significance was calculated with a one-way ANOVA test in E-H. E-H bars represent mean ± SD.

MondoA localization and transcriptional function is regulated by PPP in HEK293T cells.

A. Immunofluorescence of MondoA in HEK293T cells under high glucose media (HG) and starvation (S) conditions supplemented with the PPP inhibitor 6-aminonicotinamide (6-AN) 100 μM. Scale bars, 20 μm. B. Quantification of MondoA nuclear localization in HEK293T cells treated with 6-AN 16 and 100 μM under HG and S conditions (N=3). C. Relative mRNA levels of MondoA in HEK293T cells treated with 6-AN 16 and 100 μM under HG and S conditions measured by qPCR (N=3). D. Relative mRNA levels of MondoA downstream target TXNIP in HEK293T cells treated with 6-AN 16 and 100 μM under HG and S conditions measured by qPCR (N=3). Statistical significance was calculated with one-way ANOVA in B-D. B bars represent mean ± SD, C-D bars represent mean ± SEM. E. Working model of MML-1 regulation by hexokinases. MML-1 is positively regulated by HXK-1 and HXK-2 through different downstream mechanisms. Inhibition of the mitochondrial β-oxidation rescues MML-1 nuclear localization upon hxk-1i, while decreasing enzymes from the PPP rescues MML-1 localization upon hxk-2i. These upstream mechanisms regulate MML-1 subcellular localization between LD-mitochondria and the nucleus, which is required for the lifespan extension mediated by germ-line loss. PPP, pentose phosphate pathway; LD, lipid droplet.

Hexokinases regulate MML-1 nuclear localization and transcriptional function.

A. MML-1 nuclear localization in the fosmid line wgIs198 was quantified in intestinal cells of day one adult worms using upon hxk-1 and hxk-2 knockdown (N=3). B. Quantification of MML-1 nuclear localization in intestinal cells of day one adult wildtype and glp-1(e2141) worms supplemented with the glycolytic inhibitor 3-bromopyruvate (3-BrP) 5, 10, and 20 mM egg-on (N=2). C. Quantification of MML-1 nuclear localization in intestinal cells of day one adult glp-1(e2141) worms supplemented with the glycolytic inhibitor 2-deoxy-glucose (2-DG) 50 mM egg-on (N=2). D-E. Representative Western blot (D) and densitometry analysis (E) of hexokinases basal levels under hxk-1i, hxk-2i, hxk-3i, and mml-1i. Protein levels were normalized to actin as a loading control (N=3). F-H. Measurements of body size area (F), length (G), and pharyngeal pumping rate (H) of day one adult worms grown under hexokinase knockdown (N=3). I. Motility assay of day one adult worms grown under hexokinase knockdown (N=2). J. Relative mml-1 mRNA levels under hexokinase knockdown measured by qPCR (N=3). K. Relative mRNA expression of MML-1 downstream targets lgg-2, swt-1, and fat-5 measured by qPCR (N=3). L. Quantification of MML-1 nuclear localization in wildtype and daf-2(e1370) mutants upon luci, hxk-1i, and hxk-2i in day one adult worms (N=3). M. Lifespan of wildtype and daf-2(e1370) upon hxk-1 and hxk-2 knockdown (N=2). N. Quantification of MML-1 nuclear localization in wildtype and raga-1(ok701) mutants in day one adult worms (N=3). O. Lifespan of wildtype, raga-1(ok701), mml-1(ok849) and raga-1 mml-1 double mutant (N=3). P. Lifespan of wildtype and raga-1(ok701) upon hxk-1 and hxk-2 knockdown (N=3). Statistical significance was calculated with t-test in C and N, one-way ANOVA in A-B and E-L, and Log-rank (Mantel-Cox) test in M and O-P. A-C, F-J, L, and N bars represent mean ± SD, and E and K bars represent mean ± SEM.

MML-1 regulation by glucose metabolism is independent of mitochondrial and oxidative stress response.

A. Localization of HXK-3::mKate2 isozyme. The primary tissues are identified with arrows. Scale bars, 20 μm. B. Representative confocal images of day one adult worms expressing MML-1::GFP and the lysosome/endosome reporter LMP-1::mKate2 under luci and hxk-2i. Scale bars, 10 μm. C. Intensity plots of the ROIs from B (n>10 MML-1 positive puncta per worm). D. Quantification of mitochondrial stress response hsp-6p::GFP transcriptional reporter upon knockdown of candidate genes regulating MML-1 nuclear localization. Worms were imaged on day one of adulthood. As a positive control for induction of mitochondrial stress, worms were supplemented with antimycin A (AA) 10 μg/mL for 24 h before imaging (N=3). E. Quantification of MML-1::GFP nuclear localization of worms grown under luci and hxk-2i supplemented with AA at the L4 stage, and nuclear localization was measured after 24 h (N=3). F. Quantification of day one adult worms expressing gst-4p::GFP transcriptional reporter exposed to paraquat for 6 h (N=3). G. Quantification of MML-1::GFP nuclear localization of day one adult worms exposed to paraquat for 6 h (N=3). H. Neutral lipid content was measured on day one of adulthood by feeding worms with C1-BODIPY-C12 egg-on (N=3). I. Quantification of day one adult worms expressing the transcriptional reporter acs-2p::GFP under hexokinase knockdown (N=3). Statistical significance was calculated with one-way ANOVA in D-G. D-G bars represent mean ± SD.

HXK-1 and HXK-2 regulate MML-1 through parallel or independent pathways.

A. MML-1 nuclear localization of day one adult worms upon double knockdown of hxk-1 with enzymes of the PPP (N=3). B-D. Relative mRNA levels of MML-1 downstream targets lgg-2 (B), swt-1 (C), and fat-5 (D) in young adult worms with double knockdown of hxk-2 with enzymes of the PPP measured by qPCR (N=3). E-H. Relative mRNA levels of MML-1 downstream targets mdl-1 (E), lgg-2 (F), swt-1 (G), and fat-5 (H) in young adult worms with double knockdown of hxk-1 with enzymes of the PPP measured by qPCR (N=3). I. Quantification of G6P levels relative to total protein measured in day one adult glp-1(e2141) worms grown under hxk-1i in combination with enzymes from the PPP (N=4). J. Lifespan analysis of glp-1(e2141) with hxk-2i and T25B9.9i single and double knockdowns. Lifespan was performed in the same experiment as Figure 3I; therefore, control lifespans are shared between plots (N=3). Statistical significance was calculated with a one-way ANOVA test in A-I, and Log-rank (Mantel-Cox) test in J. A bars represent mean ± SD, B-I bars represent mean ± SEM.

MML-1 interactome is enriched with proteins from different organelles.

A. Quantified proteins in each biological replicate of wildtype and mml-1::3xFLAG anti-FLAG immunoprecipitation (IP). B. Principal component analysis (PCA) with each biological replicate of the genotypes from proteomics. C. Top candidates co-enriched in MML-1::FLAG IP grouped by predicted subcellular localization (uniport.org). D. Representative images of day one adult worms expressing the LD proteins DHS-3::GFP and PLIN-1::mCherry reporters grown with the LD dye LipiBlue egg-on. Scale bars, 10 μm. E-F. Intensity plots of the marked ROI from D showing DHS-3::GFP (E) and PLIN-1::mCherry (F) colocalization with LipiBlue. G. Expression of the ATGL-1::GFP translational reporter measured on day one adult worms grown under T25B9.9 and tkt-1 RNAi egg-on (N=3). H. Quantification of LD reporter PLIN-1::mCherry intensity in day one adult worms upon knockdown of hxk-2 and tkt-1 (N=3). I. Quantification of neutral lipid content in day one adult worms supplemented with C1-BODIPY-C12 egg-on under dgat-2 and atgl-1 knockdown (N=3). Statistical significance was calculated with a one-way ANOVA test in G-I. G-I bars represent mean ± SD.

Inhibition of PPP NADPH-producing enzymes by 6-AN induces MondoA downstream targets.

A. Experimental design for inhibition of the PPP in HEK293T cells. Cells were grown in high glucose media (HG) supplemented with 6-AN for 24 h, and then the media was replaced with either HG or starvation (S) media for 6 h. Cells were collected and used for immunostaining, RNA extraction, and metabolite quantification. B. Quantification of NADP+ and NADPH levels in HEK293T cells treated with 6-AN 16 and 100 μM under HG and S conditions (N=3). C. Relative mRNA levels of MondoA downstream target ARRDC4 in HEK293T cells treated with 6-AN 16 and 100 μM under HG and S conditions measured by qPCR (N=3). Statistical significance was calculated with one-way ANOVA in B-C. B-C bars represent mean ± SEM.