Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Sep 27;15(1):8301.
doi: 10.1038/s41467-024-52538-5.

Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis

Katherine Labbé #  1 , Lauren LeBon #  1 , Bryan King #  1 , Ngoc Vu  1 , Emily H Stoops  1 , Nina Ly  1 , Austin E Y T Lefebvre  1 , Phillip Seitzer  1 , Swathi Krishnan  1 , Jin-Mi Heo  1 , Bryson Bennett  2 , Carmela Sidrauski  3
Affiliations

Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis

Katherine Labbé et al. Nat Commun. .

Abstract

The integrated stress response (ISR) enables cells to cope with a variety of insults, but its specific contribution to downstream cellular outputs remains unclear. Using a synthetic tool, we selectively activate the ISR without co-activation of parallel pathways and define the resulting cellular state with multi-omics profiling. We identify time- and dose-dependent gene expression modules, with ATF4 driving only a small but sensitive subgroup that includes amino acid metabolic enzymes. This ATF4 response affects cellular bioenergetics, rerouting carbon utilization towards amino acid production and away from the tricarboxylic acid cycle and fatty acid synthesis. We also find an ATF4-independent reorganization of the lipidome that promotes DGAT-dependent triglyceride synthesis and accumulation of lipid droplets. While DGAT1 is the main driver of lipid droplet biogenesis, DGAT2 plays an essential role in buffering stress and maintaining cell survival. Together, we demonstrate the sufficiency of the ISR in promoting a previously unappreciated metabolic state.

PubMed Disclaimer

Conflict of interest statement

K.L., L.L., BK., N.V., E.H.S., N.L., A.E.Y.T.L., P.S., B.B., and C.S. are employees of Calico Life Sciences LLC and declare no other competing interests. C.S. is an inventor on U.S. Patent 9708247 describing ISRIB and its analogs. Rights to the invention have been licensed to Calico Life Sciences LLC from the University of California, San Francisco.

Figures

Fig. 1
Fig. 1. Dimerizable PERK enables tunable and selective control of the ISR.
a Schematic illustrating the inputs and outputs of the ISR. The ISR incorporates input from various cellular stress sensors to increase the levels of p-eIF2, the central input into the pathway. Its major outputs include a reduction in protein synthesis, the formation of SGs, and the induction of a specialized gene expression program that is in part mediated by the translational induction of the transcription factor ATF4. b Schematic illustrating the pharmacogenetic Dmr-PERK tool. Upon addition of a small molecule dimerizer, Dmr-PERK phosphorylates eIF2 and activates the ISR. c AlphaLISA measurement of the ratio of p-eIF2/eIF2 in Dmr-PERK U2OS cells following 2 h of dimerizer treatment at the indicated concentrations. Error bars show mean ± SD of three technical replicates. d Quantification of OP-Puromycin incorporation (left axis) and G3BP1-positive SGs (right axis) co-labeled in Dmr-PERK cells following 4 h of dimerizer treatment at the indicated concentration. Error bars depict mean ± SD of three technical replicates e Heatmap of protein coding gene expression in untreated (UT) Dmr-PERK cells or in cells treated with dimerizer (Dmr, 0.2 nM), thapsigargin (Tg, 100 nM), or arsenite (Ars, 0.05 mM), for the indicated amount of time. Columns show the mean expression of three replicates relative to the mean expression at t = 0 on a log2 scale. f Heatmap of genes described in e that have a significantly different time-dependent response between dimerizer (Dmr) and thapsigargin (Tg) treatments. Selected GO terms with significant enrichment by ORA within the groups of Tg-responsive genes are indicated. g Heatmap of genes described in e that have a significantly different time-dependent response between dimerizer (Dmr) and arsenite (Ars) treatments. Selected GO terms with significant enrichment by ORA within the groups of arsenite-responsive genes are indicated. a, b are created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Varying the strength and duration of ISR activation reveals distinct transcriptional responses.
a Heatmap of protein coding genes with time-dependent expression changes in Dmr-PERK cells treated with the indicated concentration of dimerizer for the indicated amount of time. Columns show the mean expression of three technical replicates relative to the mean expression at t = 0 on a log2 scale. Classes describe groups of genes clustered by their dose- and time-dependent responses b Selected GO terms with significant enrichment by ORA of the genes within each class. There were no significant enriched pathways among Class 3 genes, but the PERK-mediated unfolded protein response term identified transcripts exhibiting unique behavior. c Plot of log2 transcripts per million (TPM) over time at the indicated doses of dimerizer for genes representative of the classes described in a. GOT1 is a representative Class 1 gene, IL4I1 is a representative Class 2 gene, IL1RL1 is a representative Class 3 gene, and PCDH18 is a representative Class 4 gene.
Fig. 3
Fig. 3. ATF4 drives a metabolic gene expression signature.
a Heatmap of protein coding gene expression changes in WT and ATF4 KO Dmr-PERK cells at the indicated time points following treatment with dimerizer (0.2 nM). Genes shown have a significantly altered time course between the two genotypes. Columns show the mean expression of three technical replicates relative to the mean expression at t = 0 on a log2 scale. Color bar indicates the class of each transcript based on the dose response study in Fig. 2. Gray boxes indicate genes that did not meet statistical significance in the dose response study from Fig. 2. b Selected GO terms with significant enrichment by ORA for the upregulated ATF4-dependent genes. Top 20 GO terms are in Supplementary Data 3.
Fig. 4
Fig. 4. ISR activation rewires central carbon metabolism.
a Heatmap of significant metabolites (FDR q < 0.01) as measured by LC-MS at the indicated time points following addition of dimerizer (0.05 nM) to WT or ATF4 KO Dmr-PERK cells. Relative abundances are shown as row mean centered z-scores. b Heatmap of TCA cycle metabolites from a. c Schematic of incorporation of 13C-label from U-13C6-glucose into the TCA cycle and in the synthesis of fatty acids. d Bar plots representing fractional labeling of TCA cycle metabolites from U-13C6-glucose in WT and ATF4 KO cells treated with vehicle (Veh.) or dimerizer (Dmr, 0.05 nM) for 24 h. Error bars depict mean ± SD of three technical replicates. e Schematic of incorporation of 13C-label from U-13C5-glutamine from oxidation (blue) vs. reductive carboxylation (red). Bar plots representing fraction labeling of malate, Asn or Asp from oxidative (M + 4) (f) or reductive (M + 3) (g) metabolism of U-13C5-glutamine in WT and ATF4 KO cells treated with vehicle (Veh.) or dimerizer (Dmr, 0.05 nM) for 24 h. Error bars depict mean ± SD of three technical replicates. Bar plots representing the fraction of palmitate carbons labeled from U-13C5-glutamine (h) or U-13C6-glucose (i) in lipids from WT or ATF4 KO Dmr-PERK cells treated with vehicle (Veh.) or dimerizer (Dmr, 0.05 nM) for 24 h. Error bars depict mean ± SD of three technical replicates. Statistical significance was evaluated by two-way ANOVA followed by Tukey’s HSD test. Source data are provided as a Source Data file.
Fig. 5
Fig. 5. The ISR alters the cellular lipidome and drives LD formation to promote cell survival during stress.
a Heatmap of lipid abundances in Dmr-PERK cells treated for the indicated amount of time with dimerizer (0.05 nM). Columns show three technical replicate measurements for each time point, grouped by lipid class. Measurements are normalized to the mean abundance at t = 0 on a log2 scale. Color bars indicate triglycerides (TG), cholesterol esters (CE), diacylglycerides (DG), glycerophospholipids (GPL), plasmalogens, sphingolipids and other lipid species. b Schematic of the lipid synthesis pathways that supply the constituents of lipid droplets. Enzymes mediating each step are labeled in gray. c BODIPY labeled Dmr-PERK cells treated for 8 h with vehicle (Veh) or dimerizer (Dmr, 1 nM). Scale bar = 5 μm d Quantification of the number of LD per cell in Dmr-PERK cells treated for 8 h with vehicle (Veh.) or the indicated concentration of dimerizer (Dmr). Median with interquartile range is indicated. 240 cells were quantified per condition. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparison test. e Quantification of the number of LD in Dmr-PERK cells at the indicated time points following treatment with the indicated concentration of dimerizer. Error bars show mean ± SD per well across three independent experiments. f, g Bar plots representing the mean number of LD per cell per well in Dmr-PERK cells treated for 8 h with dimerizer (Dmr, 1 nM), ACATi (10 μM), DGAT1/2i (20 μM each) or ACCi (2 μg/mL) as indicated. Error bars show mean ± SD across three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparison test. h Quantification of the percent confluence of Dmr-PERK cells treated with vehicle, dimerizer (Dmr, 0.024 nM), ACATi (10 μM), DGAT1/2i (20 μM each) or ACCi (2 μg/mL), as indicated. Error bars show mean ± SD of two technical replicates and is representative of three independent experiments. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. DGAT1 and DGAT2 play distinct roles in ISR-induced LD formation and cell survival.
a, b Bar plots representing the mean number of LD per cell per well in Dmr-PERK cells treated for 8 h with dimerizer (Dmr, 1 nM), DGAT1i (20 μM) or DGAT2i (20 μM) as indicated. Error bars show mean ± SD across three independent experiments. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparison test. c Quantification of the percent confluence of Dmr-PERK cells treated with vehicle, dimerizer (Dmr, 0.024 nM), DGAT1i (20 μM) or DGAT2i (20 μM), as indicated. Error bars show mean ± SD of two replicates and is representative of three independent experiments. d Western blot for cleaved caspase-3 in lysates from Dmr-PERK cells treated with the indicated dose of dimerizer (Dmr), DGAT1i (20 μM) or DGAT2i (20 μM), as indicated. Quantification of the number of LD (e) and cell number (f) simultaneously imaged by high-content live-cell microscopy in Dmr-PERK cells treated with vehicle, dimerizer (Dmr), DGAT1i (20 μM) or DGAT2i (20 μM), as indicated. Error bars show mean ± SD of three independent experiments. g Western blot for ATF4 in lysates from Dmr-PERK cells treated for 24 h with dimerizer (Dmr, 0.05 nM), DGAT1i (20 μM) or DGAT2i (20 μM), as indicated. h Average z-core of ISR GeneCLIC genes calculated from nCounter gene expression profiling of Dmr-PERK cells treated for 24 h with dimerizer (Dmr, 0.05 nM), DGAT1i (20 μM) or DGAT2i (20 μM), as indicated. Error bars show mean ± SD of three technical replicates. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s multiple comparison test. Source data are provided as a Source Data file.
Fig. 7
Fig. 7. Model of ATF4-dependent and ATF4-independent ISR metabolic outputs.
The ISR increases glutathione and serine synthesis, while diverting glucose and glutamine carbon away from oxidation in mitochondria. Synthesis of aspartate, asparagine and acyl-CoA is maintained via reductive carboxylation of glutamine. These ATF4-dependent changes are accompanied by ATF4-independent inhibition of glycolysis, increased pentose phosphate pathway activity, and increased TG production and accumulation in lipid droplets. Changes in pathway utilization, metabolite abundance and enzyme expression are indicated by color (red = up, blue = down).
See this image and copyright information in PMC

References

    1. Pakos-Zebrucka, K. et al. The integrated stress response. EMBO Rep.17, 1374–1395 (2016). - PMC - PubMed
    1. Harding, H. P. et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell6, 1099–1108 (2000). - PubMed
    1. Donnelly, N., Gorman, A. M., Gupta, S. & Samali, A. The eIF2α kinases: their structures and functions. Cell. Mol. Life Sci.70, 3493–3511 (2013). - PMC - PubMed
    1. Merrick, W. C. & Pavitt, G. D. Protein synthesis initiation in eukaryotic cells. Cold Spring Harb. Perspect. Biol.10, a033092 (2018). - PMC - PubMed
    1. Hinnebusch, A. G. & Lorsch, J. R. The mechanism of eukaryotic translation initiation: new insights and challenges. Cold Spring Harb. Perspect. Biol.4, a011544 (2012). - PMC - PubMed

MeSH terms

LinkOut - more resources