Irg1 deficiency exacerbates hepatic lipid accumulation during sepsis.

(A) Oil red O staining of liver sections of WT and Irg1 KO control and cecal slurry (CS) injected mice (5μl/kg). Images are representative of 5 independent experiments. (B) Hepatic triglyceride content. n=7 mice per group. (C) Oleate-loaded primary hepatocytes (top panel) and AML12 cells (bottom panel) treated with vehicle (DMSO) or 4-OI (250µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. * denotes p<0.05, *** p<0.001. Scale bars are 50μm.

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 hours. 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 hours. Quantification on the right. n=4 independent experiments. (D) Western blot of liver lysates of female LPS injected WT and Irg1 KO mice. Quantification on the right. n=5 mice per group. (E) Nile Red images of lipid-loaded hepatocytes treated with 4-OI (250µM) ± etomoxir (4µM). n=3 independent experiments. * denotes p<0.05, ** p<0.01, *** p<0.001.

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 hours, 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 hours followed by stimulation with MG132 for 6 hours. 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 hours. ** denotes p<0.01

Irg1 deficiency promotes hypothermia and BAT dysfunction during endotoxin challenge.

(A) Core body temperature in female WT and Irg1 KO mice following LPS injection (5mg/kg). n=5-8 mice per group. (B) Western blot of UCP1, PGC-1α, and GAPDH in BAT of LPS injected WT and Irg1 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 Irg1 KO mice. n=5-8 mice per group. (E) BAT weight 24h post-LPS injection in WT and Irg1 KO mice. n=5-8 mice per group. * denotes p<0.05, ** p<0.01.

Irg1 deficiency impairs systemic substrate utilization during sepsis.

(A) Energy Expenditure (kcal/hr/kg) during baseline and (C) post-LPS injection in female Irg1 KO and WT controls. Plots represent 24-hour cycle. n=3 mice per group. (B,D) Area under the curve for energy expenditure values over 24-hour cycle from panel B and D. (E) Respiratory exchange ratio (RER) during baseline and (G) post-LPS injection in female Irg1 KO and WT littermate controls. Plots represent 24-hour cycle. n=3 mice per group. (F,H) Area under the curve for RER values over 24-hour cycle from panel F and H. Statistical significance was calculated using an unpaired 2-tailed Student’s t test.