Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation
- Department of Pathology, Section on Comparative Medicine, Wake Forest School of Medicine, United States
- Department of Internal Medicine, Section on Molecular Medicine, Wake Forest School of Medicine, United States
Figures
Figure 1 with 1 supplement
Aconitase decarboxylase 1 (Acod1) deficiency exacerbates hepatic lipid accumulation during sepsis.
(A) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. (B) Hepatic triglyceride content. n=7 mice per group. (C) Oleate-loaded primary hepatocytes (top panel) and AML12 cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.
Figure 1—figure supplement 1
BODIPY staining of frozen liver section of wild-type (WT) and Acod1-/- KO livers stained 24 hr post-LPS injection. Scale bars are 50 μm.
Figure 2
4-octyl itaconate (4-OI) promotes mitochondrial fatty acid uptake and clearance.
(A) Pathway analysis of significantly altered proteins from global proteomics of AML12 cells stimulated with 4-OI for 24 hr. n=5 biological replicates per group. (B) Quantification of CPT1a/CPT2 in proteomics analysis. (C) Western blot of CPT1a, CPT2, and SLC25a20 in AML12 stimulated with 4-OI for 24 hr. Quantification on the right. n=4 independent experiments. (D) Western blot of liver lysates of female LPS injected wild-type (WT) and aconitase decarboxylase 1 (Acod1) KO mice. Quantification on the right. n=5 mice per group. (E) Nile Red images of lipid-loaded hepatocytes treated with 4-OI (250 µM) ± etomoxir (4 µM). n=3 independent experiments. *p<0.05, **p<0.01, ***p<0.001. Scale bars are 50 μm.
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Figure 2—source data 1
Source file for Western blot 2c.
- https://cdn.elifesciences.org/articles/92420/elife-92420-fig2-data1-v1.pdf
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Figure 2—source data 2
Source file for Western blot 2d.
- https://cdn.elifesciences.org/articles/92420/elife-92420-fig2-data2-v1.pdf
Figure 3
4-octyl itaconate (4-OI) stabilizes CPT1a protein expression.
(A) Western blot and quantification of CPT1a in AML12 cells that were pretreated with vehicle or 4-OI for 24 hr, then stimulated with cycloheximide (CHX). Quantification on the right. n=3 independent experiments. (B) Immunoprecipitation (IP) of CPT1a in AML12 cells pretreated with 4-OI for 24 hr followed by stimulation with MG132 for 6 hr. Equal amounts of proteins were IP’d with anti-CPT1a and subjected to Western blot analysis with ubiquitin antibody. 5% input below. n=3 independent experiments. (C) In-gel fluorescence of rhodamine in iTalk labeled Raw macrophages and AML12 hepatocytes. (D) Pathway analysis of global proteomics of iTalk-enriched proteins in AML12 cells stimulated with ITalk for 4 hr. **p<0.01.
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Figure 3—source data 1
Source file for Western blot 3a.
- https://cdn.elifesciences.org/articles/92420/elife-92420-fig3-data1-v1.pdf
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Figure 3—source data 2
Source file for Western blot 3b.
- https://cdn.elifesciences.org/articles/92420/elife-92420-fig3-data2-v1.pdf
Figure 4
Aconitase decarboxylase 1 (Acod1) deficiency promotes hypothermia and brown adipose tissue (BAT) dysfunction during endotoxin challenge.
(A) Core body temperature in female wild-type (WT) and Acod1 KO mice following LPS injection (5 mg/kg). n=5–8 mice per group. (B) Western blot of UCP1, PGC-1α, and GAPDH in BAT of LPS injected WT and Acod1 KO mice. (C) Quantification of UCP1 protein normalized to GAPDH. n=5–8 mice per group. (D) qPCR of UCP1 in BAT of LPS injected WT and Acod1 KO mice. n=5–8 mice per group. (E) BAT weight 24 hr post-LPS injection in WT and Acod1 KO mice. n=5–8 mice per group. *p<0.05, **p<0.01.
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Figure 4—source data 1
Source file for Western blot 4b.
- https://cdn.elifesciences.org/articles/92420/elife-92420-fig4-data1-v1.pdf
Figure 5
Aconitase decarboxylase 1 (Acod1) deficiency impairs systemic substrate utilization during sepsis.
(A) Energy Expenditure (kcal/hr/kg) during baseline and (C) post-LPS injection in female Acod1 KO and wild-type (WT) controls. Plots represent 24 hr cycle. n=3 mice per group. (B, D) Area under the curve for energy expenditure values over 24 hr cycle from panel B and D. (E) Respiratory exchange ratio (RER) during baseline and (G) post-LPS injection in female Acod1 KO and WT littermate controls. Plots represent 24 hr cycle. n=3 mice per group. (F, H) Area under the curve for RER values over 24 hr cycle from panel F and H. Statistical significance was calculated using an unpaired two-tailed Student’s t-test.
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Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation
eLife 12:RP92420.
https://doi.org/10.7554/eLife.92420.2
