The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse

Communications Biology · 10 authors, 10 centres

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POPULATIONMice (Mus musculus, C57BL/J6) with systemic overexpression of SLC13A5 or SLC25A1 (inducible Tet-Off system under Rosa26 locus) and wild-type littermates

INTERVENTIONSystemic overexpression of SLC13A5 (from conception, birth, or weaning) or SLC25A1 (from conception); treatment with compound 9 (ATase1/2 inhibitor) in SLC13A5 sTg mice

COMPARISONWild-type littermates; SLC25A1 sTg vs SLC13A5 sTg; SLC13A5 sTg with vs without compound 9

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TL;DR

Mice with systemic overexpression of the citrate transporter SLC13A5 develop a severe progeria-like phenotype with premature death, whereas overexpression of SLC25A1 does not. Despite both transporters increasing cytosolic citrate and acetyl-CoA levels, SLC13A5 uniquely upregulates the ER acetylation machinery (AT-1, ATase1/2) and hyperacetylates ER cargo proteins, including ATG9A. Inhibiting ATase1/2 with compound 9 rescues the progeria-like phenotype and extends lifespan, highlighting a specific link between SLC13A5-driven citrate import, ER proteostasis, and premature aging.

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**Background:** Cytosolic citrate is imported from mitochondria by SLC25A1 and from the extracellular milieu by SLC13A5. In the cytosol, citrate is converted by ACLY to acetyl-CoA, which can be transported into the endoplasmic reticulum (ER) by SLC33A1 (AT-1). Mutations in these transporters are linked to human diseases including developmental delay, epilepsy, and progeria. This study investigates the systemic effects of overexpressing SLC25A1 or SLC13A5 in mice.

**Methods:** Transgenic mice with inducible Tet-Off systemic overexpression of SLC25A1 (SLC25A1 sTg) or SLC13A5 (SLC13A5 sTg) were generated under the Rosa26 locus. SLC25A1 sTg mice overexpressed from conception. SLC13A5 sTg mice were studied under three conditions: overexpression from conception (OC), from birth (OB), and from weaning (OW). Phenotypic characterization included body weight, lifespan, histology (skin, bone, spleen, lymph nodes, cornea), senescence markers (p16, p21, SA-β-gal), and metabolic assays (citrate, acetyl-CoA, malate in subcellular fractions). Quantitative acetyl-proteomics (stoichiometry of lysine acetylation) and lipidomics (LC-MS) were performed on liver, serum, skin, and brain. ER acetylation machinery was assessed by Western blot for AT-1, acetylated ER proteins, and ATG9A acetylation. SLC13A5 sTg mice were treated with compound 9 (C9), an inhibitor of ER acetyltransferases ATase1/2.

**Key Results:** SLC25A1 sTg mice were smaller but had normal lifespan and no progeria. SLC13A5 sTg^OC^ died perinatally; SLC13A5 sTg^OB^ and ^OW^ developed a severe progeria-like phenotype with reduced lifespan (maximum 175 days for males, 147 days for females), skin lesions, hair loss, hunched posture, rectal prolapse, splenomegaly, enlarged lymph nodes, systemic inflammation, osteoporosis, corneal changes (10-15% of mice), and increased senescence markers (p16, p21, SA-β-gal). Both models had increased cytosolic citrate and acetyl-CoA, and ER acetyl-CoA. SLC13A5 sTg mice showed a 3-fold upregulation of AT-1 and hyperacetylation of ER cargo proteins and ATG9A (approx. 4-fold), while SLC25A1 sTg did not. Acetyl-proteomics revealed 727 significantly changed lysine sites (599 proteins) in SLC13A5 sTg and 733 sites (591 proteins) in SLC25A1 sTg, with only ~30% overlap (224 sites, 193 proteins). Unique to SLC13A5 sTg were 698 sites (500 proteins) enriched in ribosomal, ER translational, and quality control pathways. Lipidomics showed 68 significantly changed lipids in SLC13A5 sTg liver vs 21 in SLC25A1 sTg, with only one common species (PC 34:6). Treatment with compound 9 reduced ER acetylation, normalized ATG9A acetylation, improved phenotype, and significantly increased lifespan (P<0.0005).

**Clinical Implications:** This study demonstrates that the source of cytosolic citrate (mitochondrial vs extracellular) determines distinct metabolic and phenotypic outcomes, with SLC13A5-driven citrate import specifically linked to ER proteostasis dysfunction and progeria. The rescue by ATase inhibition suggests a potential therapeutic target for diseases involving SLC13A5 hyperactivity or ER acetylation dysregulation. These findings highlight the importance of metabolite compartmentalization in signaling and disease, and may inform strategies for treating progeroid syndromes and related metabolic disorders.

PICO

PPOPULATION

Mice (Mus musculus, C57BL/J6) with systemic overexpression of SLC13A5 or SLC25A1 (inducible Tet-Off system under Rosa26 locus) and wild-type littermates

IINTERVENTION

Systemic overexpression of SLC13A5 (from conception, birth, or weaning) or SLC25A1 (from conception); treatment with compound 9 (ATase1/2 inhibitor) in SLC13A5 sTg mice

OOUTCOME

Phenotype (progeria-like features, lifespan, body weight, skin lesions, bone density, organ weights, senescence markers), metabolic profile (citrate, acetyl-CoA, malate levels in subcellular fractions), acetyl-proteome (stoichiometry of lysine acetylation), lipidome, ER acetylation (AT-1 expression, ATG9A acetylation), and rescue by compound 9

STUDY TYPE

other

SPECIALTY

animal science

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ORIGINAL PAPER

PMC10492862

The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse

Communications Biology

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ABSTRACT

Cytosolic citrate is imported from the mitochondria by SLC25A1, and from the extracellular milieu by SLC13A5. In the cytosol, citrate is used by ACLY to generate acetyl-CoA, which can then be exported to the endoplasmic reticulum (ER) by SLC33A1. Here, we report the generation of mice with systemic overexpression (sTg) of SLC25A1 or SLC13A5. Both animals displayed increased cytosolic levels of citrate and acetyl-CoA; however, SLC13A5 sTg mice developed a progeria-like phenotype with premature death, while SLC25A1 sTg mice did not. Analysis of the metabolic profile revealed widespread differences. Furthermore, SLC13A5 sTg mice displayed increased engagement of the ER acetylation machinery through SLC33A1, while SLC25A1 sTg mice did not. In conclusion, our findings point to different biological responses to SLC13A5- or SLC25A1-mediated import of citrate and suggest that the directionality of the citrate/acetyl-CoA pathway can transduce different signals.

CITATION EXPORT

Gonzalo Fernandez-Fuente, Katherine A. Overmyer, Alexis J. Lawton et al.. The citrate transporters SLC13A5 and SLC25A1 elicit different metabolic responses and phenotypes in the mouse. Communications Biology. (2023). https://doi.org/10.1038/s42003-023-05311-1

AUTHORS

10 · 10 CENTRES

Gonzalo Fernandez-Fuente, Katherine A. Overmyer, Alexis J. Lawton, Ildiko Kasza, Samantha L. Shapiro, Patricia Gallego-Muñoz, Joshua J. Coon, John M. Denu, Caroline M. Alexander, Luigi Puglielli

1https://ror.org/01y2jtd41grid.14003.360000 0001 2167 3675Department of Medicine, University of Wisconsin-Madison, Madison, WI USA
2https://ror.org/01y2jtd41grid.14003.360000 0001 2167 3675Waisman Center, University of Wisconsin-Madison, Madison, WI USA
3https://ror.org/01y2jtd41grid.14003.360000 0001 2167 3675Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, WI USA
4https://ror.org/05cb4rb43grid.509573.d0000 0004 0405 0937Morgridge Institute for Research, Madison, WI USA
5grid.14003.360000 0001 2167 3675Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI USA
6https://ror.org/01y2jtd41grid.14003.360000 0001 2167 3675McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI USA
7https://ror.org/01fvbaw18grid.5239.d0000 0001 2286 5329Department of Cell Biology, Genetics, Histology and Pharmacology, Faculty of Medicine, University of Valladolid, Valladolid, Spain
8https://ror.org/01y2jtd41grid.14003.360000 0001 2167 3675Department of Chemistry, University of Wisconsin-Madison, Madison, WI USA
9https://ror.org/01nh3sx96grid.511190.d0000 0004 7648 112XGeriatric Research Education Clinical Center, Veterans Affairs Medical Center, Madison, WI USA
10https://ror.org/01y2jtd41grid.14003.360000 0001 2167 3675Department of Neuroscience, University of Wisconsin-Madison, Madison, WI USA