Conserved lipid metabolic reprogramming confers hypoxic and aging resilience

Abstract

The Arctic ground squirrel (AGS, Urocitellus parryii), an extreme hibernator, exhibits remarkable resilience to stressors like hypoxia and hypothermia, making it an ideal model for studying cellular metabolic adaptation. The underlying mechanisms of AGS resilience are largely unknown. Here, we use lipidomic and metabolomic profiling to discover specific downregulation of triglyceride lipids and upregulation of the lipid biosynthetic precursor malonic acid in AGS neural stem cells (NSC) versus murine NSCs. Inhibiting lipid biosynthesis recapitulates hypoxic resilience of squirrel NSCs. Extending this model, we find that acute exposure to hypoxia downregulates key lipid biosynthetic enzymes in C. elegans, while inhibiting lipid biosynthesis reduces mitochondrial fission and facilitates hypoxic survival. Moreover, inhibiting lipid biosynthesis protects against APOE4-induced pathologies and aging trajectories in C. elegans. These findings suggest triglyceride downregulation as a conserved metabolic resilience mechanism, offering insights into protective strategies for neural tissues under hypoxic or ischemic conditions, APOE4-induced pathologies and aging.

Keywords: C. elegans; Arctic Ground Squirrel; Hypoxia; Lipid Biosynthetic Enzymes; Triglyceride Lipids.

Figure 1. Saturated and unsaturated triglycerides are downregulated in AGS.

(A) Schematic of the lipidomic analysis of mouse neural stem cells (NSCs) and Arctic ground squirrel NSCs under normal cell culture conditions. (B) Quantification of lipids in AGS NSCs and mouse NSCs showing that triglycerides (normalized to protein) were significantly downregulated in AGS NSCs compared to mouse NSCs under normal culture conditions. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. ****P < 0.0001, n.s. indicates nonsignificant. n  =  3 biological replicates. (C) Quantification of triglycerides level in AGS NSCs and mouse NSCs under normal culture conditions. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. ***P < 0.001. n  =  3 biological replicates. (D) Quantification of fatty acids in triglycerides in AGS NSCs and mouse NSCs showing that both saturated fatty acids and unsaturated fatty acids were significantly downregulated in AGS NSCs compared to mouse NSCs under normal culture conditions. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. ****P < 0.0001, ***P < 0.001. n  =  3 biological replicates. (E) Representative confocal fluorescence images displaying single mid-plane views of lipid droplets and plasma membrane staining in mouse and AGS NSCs (left). Quantification of lipid droplets per mid-plane image in mouse and AGS NSCs (right). Scale bars: 10 µm. Data were presented as means ± SEM. P values calculated by unpaired two-tailed t-tests. n.s. indicates nonsignificant (n = 30 cells per condition). (F) Quantification of metabolites in AGS NSCs and mouse NSCs showing that malonic acid was significantly upregulated in AGS NSCs compared to mouse NSCs under normal conditions. Data were presented as means ± SEM. P values calculated by unpaired two-tailed t-tests. **P < 0.01, n.s. indicates nonsignificant. n  =  3 biological replicates. (G, H) Schematic illustrating functional FAS (fatty acid synthesis) in the normal state of mouse NSCs and FAS-impaired state in AGS NSCs. Source data are available online for this figure.

Figure 2. Downregulation of unsaturated triglycerides and unsaturated lipid droplets by HIF-1 and hypoxia in C. elegans.

(A) Schematic illustrating fatty acid synthesis and desaturation pathways. (B) Representative confocal fluorescence images showing the expression of FASN-1::GFP under normal or hypoxic (0.1%) conditions for 24 h post L4 stages. Scale bars: 100 µm (low mag) or 10 µm (high mag). (C) Representative confocal fluorescence images showing high-resolution Z-stack views of fat-6p::fat-6::GFP under normal or hypoxic (0.1%) conditions for 24 h post L4 stages. Scale bars: 10 µm. (D) Representative confocal fluorescence images showing low and high-resolution Z-stack views of fat-6p::fat-6::GFP under normal or hypoxic (0.5%) conditions for 24 h post L4 stages. Scale bars: 10 µm. (E) Quantification of the percentage of animals with fat-6p::fat-6::GFP fluorescence intensities downregulated under normal or hypoxic (0.5%) conditions for 24 h post L4 stages. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. ***P < 0.001 (normal, n = 30 animals, hypoxic, n = 37 animals). (F) Representative confocal fluorescence images showing the downregulation of dhs-3p::dhs-3::GFP-labeled lipid droplet abundance and number under normal or hypoxic (0.1%) conditions for 24 h post L4 stages. Scale bars: 10 µm. (G) Representative confocal fluorescence images showing the expression of HIF-1-dependent transcriptional reporter, nis470 (cysl-2p::mVenus) abundance under DMSO or VHL-1 inhibitor VH-298 (1 mg/ml) for 96 h post embryos stage. Scale bars: 10 µm. (H) Representative confocal fluorescence images showing low and high-resolution Z-stack views of fat-6p::fat-6::GFP under DMSO or VHL-1 inhibitor VH-298 (1 mg/ml) for 96 h post embryos stage. Scale bars: 10 µm. (I) Quantification of the percentage of animals with fat-6p::fat-6::GFP fluorescence intensities downregulated under DMSO or VHL-1 inhibitor VH-298 (1 mg/ml) for 96 h post embryos stage. Data were presented as means ± SEM. P values calculated by unpaired two-tailed t-tests. *P < 0.05 (n = 30 animals per condition). (J) Representative confocal fluorescence images showing fat-6p::fat-6::GFP animals fed with control RNAi or RNAi against hif-1 or vhl-1 under normal or hypoxic conditions. Scale bars: 10 µm. (K) Representative confocal fluorescence images showing dhs-3p::dhs-3::GFP animals fed with control RNAi or RNAi against hif-1 or vhl-1 under normal conditions. Scale bars: 10 µm. (L) Representative confocal fluorescence images showing FASN-1::GFP animals fed with control RNAi or RNAi against vhl-1 under normal conditions. Scale bars: 10 µm. Source data are available online for this figure.

Figure 3. HIF activation or pharmacological inhibition of fatty acid biosynthesis reduces cellular lipid droplets and hypoxic injuries.

(A) Representative confocal microscopy images and quantification of lipid droplets (green) and plasma membrane (magenta) in HEK293T cells and a non-degradation HIF-1 ectopic expression HEK293T cell line. Scale bar: 10 μm. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. ****P < 0.0001 (n = 26 cells per condition). (B) Representative confocal microscopic images of lipid droplets (green), plasma membrane (magenta), and DAPI (blue) in mouse NSCs treated with DMSO, or 20 nM TOFA, 20 nM Cerulenin, or alpha ketoglutarate for 16 h. Scale bar: 10 μm. (C, D) Representative confocal fluorescence images of membrane staining and quantification of the percentage of membrane abnormal (data were presented as means ± S.D.) or LDH release (data were presented as means ± SEM) under DMSO or Cerulenin, for 16 h in mouse NSCs, followed by normal or acute hypoxia for 60 min. Scale bar: 10 μm. P values calculated by two-way ANOVA. ****P < 0.0001 (n > 30 cells per condition for membrane abnormal). (E, F) Representative confocal fluorescence images of membrane staining and quantification of the percentage of membrane damage under DMSO or the ACC inhibitor TOFA for 16 h in mouse NSCs, followed by normal or acute hypoxia for 60 min. Scale bar: 10 μm. Data were presented as means ± S.D. P values calculated by two-way ANOVA. ****P < 0.0001 (n > 30 cells per condition). (G, H) Representative confocal fluorescence images of membrane staining and quantification of the percentage of membrane damage under DMSO or the ACC inhibitor TOFA for 16 h in AGS NSCs, followed by normal or acute hypoxia for 60 min. Scale bar: 10 μm. Data were presented as means ± S.D. P values calculated by two-way ANOVA. ****P < 0.0001 (n > 30 cells per condition). Source data are available online for this figure.

Figure 4. Pharmacological inhibition of triglyceride biosynthesis protects against hypoxia in C. elegans.

(A) Quantification of the percentage of worm death in WT or mutant alleles fasn-1(g14) or fat-6(tm331); fat-5(tm420) under long-term hypoxia conditions for 72 h. Data were presented as means ± SEM. P values calculated by one-way ANOVA. ***P < 0.001; **P < 0.01, n.s indicates nonsignificant (n > 150 worms per condition). (B) Quantification of the percentage of worm death under DMSO or the ACC inhibitor TOFA starting at embryos for 48 h, followed by long-term hypoxia for 72 h. Data were presented as means ± SEM. P values calculated by unpaired two-tailed t-tests. *P < 0.05 (n > 150 worms per condition). (C) Representative confocal fluorescence images showing high-resolution Z-stack views of dhs-3p::dhs-3::GFP under DMSO or the ACC inhibitor TOFA for 72 h (YA) post-embryo stage. Scale bars: 10 µm or 1 µm (magnification). (D) Representative confocal fluorescence images showing high-resolution Z-stack views of mai-2::GFP-labeled mitochondrial morphology under normal or short-term hypoxic (0.5%) conditions for 24 h post-L4 stages. Scale bars: 10 µm. (E) Representative confocal fluorescence images showing high-resolution Z-stack views of mai-2::GFP-labeled mitochondrial morphology, with effect more pronounced in body wall muscles (arrows) by exposure to the ACC inhibitor TOFA for 48 h starting from the embryo stage, followed by short-term hypoxia for 24 h. Scale bars: 10 µm. (F) Quantification of the percentage of mitochondria with “total” fission in mai-2::GFP-labeled mitochondria under DMSO or the ACC inhibitor TOFA for 48 h starting from the embryo stage, followed by short-term hypoxia for 24 h. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. **P < 0.01 (n > 25 animals per condition). (G) Representative confocal fluorescence images showing high-resolution Z-stack views of mCherry-PDR-1-labeled mitochondria morphology under DMSO or the ACC inhibitor TOFA for 48 h starting from the embryo stage, followed by short-term hypoxia for 24 h. Scale bars: 10 µm. (H) Schematic model showing that the ACC inhibitor TOFA-induced downregulation of saturated and unsaturated triglyceride protects against mitochondrial fission, contributing to the alleviation of organismal death. Source data are available online for this figure.

Figure 5. Downregulation of saturated and unsaturated triglycerides by TOFA protects against APOE-induced pathologies in C. elegans.

(A) Representative low and high magnification confocal microscopic images of hsp-16.2p::GFP; unc-54p::mCherry or APOE4; hsp-16.2p::GFP; unc-54p::mCherry (white arrows) at the YA stage, treated with DMSO or 1 mg/ml TOFA starting at the embryo stage. Scale bar: 10 μm. (B) Quantification of the percentage of hsp-16.2p::GFP positive expression in the BWM of WT or APOE4 at the YA stage, treated with DMSO or 1 mg/ml TOFA starting at the embryo stage. Data were presented as means ± SEM. P values calculated by two-way ANOVA. **P < 0.01 (n > 30 animals per condition). (C) Quantification of the percentage of decrease in Q40::YFP aggregation in the BWM of WT or APOE4 at the Day3 post L4 stage, treated with DMSO or 1 mg/ml TOFA starting at the embryo stage. Data were presented as means ± SEM. P values calculated by two-way ANOVA. **P < 0.01 (n > 30 animals per condition). (D) Representative high magnification confocal microscopic images of unc-54p::Q40::YFP or APOE4; unc-54p::Q40::YFP (white arrows) at the YA stage, treated with DMSO or 1 mg/ml TOFA starting at the embryo stage. Scale bar: 10 μm. (E) Representative low and high magnification confocal microscopic images of unc-54p::Q40::YFP or APOE4; unc-54p::Q40::YFP (white arrows) at the day 3 post L4 stage, treated with DMSO or 1 mg/ml TOFA starting at the embryo stage. Scale bar: 100 μm (low mag) or 10 μm (high mag). (F) Lifespan curves of WT or APOE4 animals treated with DMSO or TOFA starting at the embryo stage, followed by survival counting starting at L4 at 25 °C. Data were presented as means without error bars. The lifespan assay was quantified using Kaplan–Meier lifespan analysis, and P values were calculated using the log-rank test. **P < 0.01 (n > 40 animals per condition). (G) Representative confocal microscopic images of PVD neurons (wyIs592[ser-2prom-3p::myr-GFP]) in pan-neuronal expression of APOE4(vxIs824) animals at the young adult stage on NGM treated with DMSO or TOFA starting at the embryo stage, showing that TOFA rescues APOE4-induced PVD abnormalities with an apparent loss of the third and fourth branches. Solid and open triangles to indicate the presence and absence of PVD neurites, respectively. Scale bar: 10 μm. (H) Quantification of the percentage of PVD neurons that are abnormal (with the third and fourth branches of PVD neurons missing or severed) in pan-neuronal APOE4 animals at the young adult stage on NGM treated with DMSO or TOFA starting at the embryo stage. Data were presented as means ± SEM. P values calculated by two-way ANOVA. **P < 0.01 (n > 30 animals per condition). Source data are available online for this figure.

Figure 6. Downregulation of saturated and unsaturated triglyceride protect against aging in C. elegans.

(A) Representative confocal fluorescence images showing high-resolution Z-stack views of fat-6p::fat-6::GFP under conditions of 7 days at T25, 7 days at T20, or 7 days at T15 post day 1 stage. Scale bars: 100 µm or 10 µm (magnification). (B) Representative confocal fluorescence images showing high-resolution Z-stack views of dhs-3p::dhs-3::GFP under conditions of 7 days at T25, 7 days at T20, or 7 days at T15 post day 1 stage. Scale bars: 100 µm or 10 µm (magnification). (C) Quantification of the percentage of animals with fat-6p::fat-6::GFP fluorescence intensities downregulated under conditions of 7 days at T25, 7 days at T20, or 7 days at T15 post day 1 stage. Data were presented as means ± S.D. P values calculated by two-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001 (n > 25 animals per condition). (D) Quantification of the percentage of animals with dhs-3p::dhs-3::GFP fluorescence intensities downregulated under conditions of 7 days at T25, 7 days at T20, or 7 days at T15 post day 1 stage. Data were presented as means ± S.D. P values calculated by two-way ANOVA. *P < 0.05, ***P < 0.001, ****P < 0.0001, n.s indicates nonsignificant (n > 25 animals per condition). (E) Lifespan curves of WT animals treated with DMSO or TOFA starting at the embryo stage, followed by survival counting starting at L4 at 25 °C. Data were presented as means without error bars. The lifespan assay was quantified using Kaplan–Meier lifespan analysis, and P values were calculated using the log-rank test. ****P < 0.0001 (n > 40 animals per condition). (F) Lifespan curves of WT animals treated with DMSO or TOFA starting at the embryo stage, followed by survival counting starting at L4 at 20 °C. Data were presented as means without error bars. The lifespan assay was quantified using Kaplan–Meier lifespan analysis, and P values were calculated using the log-rank test. ****P < 0.0001 (n > 40 animals per condition). (G) Lifespan curves of WT animals treated with DMSO or TOFA starting at the embryo stage, followed by survival counting starting at L4 at 15 °C. Data were presented as means without error bars. The lifespan assay was quantified using Kaplan–Meier lifespan analysis, and P values were calculated using the log-rank test. ****P < 0.0001 (n > 40 animals per condition). Source data are available online for this figure.

Figure 7. Model of triglyceride downregulation in protecting against hypoxic injury, APOE4-induced pathologies, and aging.

(A) HIF-1 suppresses fatty acid biosynthesis and desaturation, leading to the downregulation of triglycerides to protect against hypoxia, APOE4 pathologies, and aging trajectories in C. elegans. Pharmacologically inhibiting TAG biosynthesis with TOFA can largely recapitulate these effects in HIF-1 activation in C. elegans. (B, C) Normal fatty acid biosynthesis and desaturation in mouse neural cells increase triglycerides, which can promote hypoxia sensitivity and injuries. In contrast, reduced triglyceride biosynthesis in AGS neural cells can contribute to hypoxia resilience. Other important players, including mitochondria and ROS, are omitted for clarity.

Figure EV1. Characterization of lipid droplets in AGS NSCs and mouse NSCs under normal conditions.

(A) Quantification of the percentage of cells with varying numbers of lipid droplets per mid-plane image in mouse and AGS NSCs (n > 25 cells per condition).

Figure EV2. Characterization of expression patterns of FAT-6 and lipid droplets with various conditions in C. elegans.

(A) Representative confocal fluorescence images showing the expression of an integrated transgene fat-6p::fat-6::GFP under normal conditions at L1-L4 stages. Scale bars: 100 µm. (B) Representative Low and high magnification confocal fluorescence images showing the expression of fat-6p::fat-6::GFP under normal conditions L4 stages. Scale bars: 10 µm. (C) Representative confocal fluorescence images showing low and high-resolution Z-stack views of fat-6p::fat-6::GFP under normal or hypoxic (5%) conditions for 24 h post L4 stages. Scale bars: 10 µm. (D) Quantification of the percentage of animals with fat-6p::fat-6::GFP fluorescence intensities downregulated under normal or hypoxic (5%) conditions for 24 h post L4 stages. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. n.s indicates nonsignificant (n > 25 animals per condition). (E) Representative confocal fluorescence images showing fat-6p::fat-6::GFP fed with control RNAi or RNAi against hif-1. Scale bars: 10 µm. (F) Representative confocal fluorescence images showing the downregulation of dhs-3p::dhs-3::GFP-labeled lipid droplet number and size under normal or hypoxic (0.5%) conditions for 24 h post-L4 stages. Scale bars: 10 µm. (G) Schematic diagram of HIF-1, VHL-1, and the drug VH-298. Representative confocal fluorescence images showing the upregulation of HIF-1 target reporter cysl-2p::Venus treat with DMSO or VHL-1 inhibitor VH-298 (1 mg/ml) for 48 h starting at embryos. Scale bars: 10 µm. (H) Representative confocal fluorescence images showing the upregulation of HIF-1 target reporter cysl-2p::Venus treat with DMSO or VHL-1 inhibitor VH-298 (10 mg/ml) for 48 h starting at embryos. Scale bars: 10 µm. (I) Representative confocal fluorescence images showing the upregulation of HIF-1 target reporter cysl-2p::Venus treat with DMSO or VHL-1 inhibitor VH-298 (10 mg/ml) for 96 h starting at embryos. Scale bars: 10 µm.

Figure EV3. Characterization of TOFA in mouse neural stem cell.

(A, B) Schematic of FASN inhibitor cerulenin and acetyl-CoA carboxylase (ACC) inhibitor TOFA (5-(Tetradecyloxy)-2-furoic acid), which blocks both saturated and unsaturated triglyceride biosynthesis. (C, D) Representative images and LDH release of mouse NSCs treated with different concentrations of TOFA or DMSO for 16 h. Data were presented as means ± SEM. P values calculated by one-way ANOVA. ****P < 0.0001, n.s indicates nonsignificant. n  =  3 biological replicates. Scale bar: 100 μm.

Figure EV4. Characterization of TOFA and expression patterns of mCherry::PDR-1 upon exposure to short-term hypoxia in C. elegans.

(A) Representative images of body size in wild-type N2 animals treated with varying concentrations of DMSO or TOFA, starting at the embryo stage at 20 °C. Scale bar: 100 μm. (B) Representative confocal fluorescence images and quantification of the percentage of animals with fat-6p::fat-6::GFP fluorescence intensities downregulated treated with DMSO or 1 mg/ml TOFA starting at the embryo stage. Scale bar: 10 μm. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. **P < 0.01 (n = 30 animals per condition). (C) Representative confocal fluorescence images showing high-resolution Z-stack tail area views of mCherry-PDR-1-labeled mitochondria morphology under normal or hypoxia (0.5%) for 24 h post-L4 stages. Scale bars: 10 µm.

Figure EV5. TOFA cannot rescue APOE4-induced neuronal lysosomal abnormalities in C. elegans.

(A) Representative high magnification confocal microscopic images of neuronal specific lysosomal membrane reporter (white arrows) ceIs56 [unc-129p::ctns-1::mCherry + nlp-21p::Venus + ttx-3p::RFP]) at the YA stage, treated with DMSO or TOFA starting at the embryo stage. Scale bar: 1 μm.

Figure EV6. FAT-6::GFP downregulation during starvation and aging in C. elegans.

(A) Representative confocal fluorescence images showing high-resolution Z-stack views of fat-6p::fat-6::GFP under 20 °C normal conditions or starvation of L1-L2 stages. Scale bars: 100 µm. (B) Quantification of the percentage of animals with fat-6p::fat-6::GFP fluorescence intensities downregulated under normal or starvation conditions. Data were presented as means ± S.D. P values calculated by unpaired two-tailed t-tests. ****P < 0.0001 (n > 25 animals per condition). (C) Representative confocal fluorescence images showing low-resolution Z-stack views of fat-6p::fat-6::GFP under 20 °C normal conditions for 0 day, 1 day, 3 days or 7 days post L4 stages. Scale bars: 100 µm. (D) Representative confocal fluorescence images showing low and high-resolution Z-stack views of FASN-1::GFP under 20 °C normal conditions or starvation (white arrows indicate positively changed worms). Scale bars: 100 µm or 10 µm (magnification). (E) Representative confocal fluorescence images showing low and high-resolution Z-stack views of DHS-3::GFP under 20 °C normal conditions or starvation. Scale bars: 100 µm or 10 µm (magnification).