Extended Data Fig. 9: Functions of peroxisomal β-oxidation and ROS in regulating ATGL levels in liver.
From: Peroxisomal β-oxidation acts as a sensor for intracellular fatty acids and regulates lipolysis
(a-b) Pex2 transcript in the liver and adipose tissue of liver-specific Pex2 knockout mice (Pex2KO) was quantified by qPCR (N = 5). (c) Atgl transcript in Pex2 knockout liver was quantified by qPCR (WT N = 7; Pex2KO N = 9). (d) ATGL lipase activity was measured using the liver lysate from Pex2KO mice (N = 7). (e) Hepatic peroxisomes were extracted from the liver-specific Cat knockout mice to determine cysteine sulfenic acid modification levels of peroxisomal proteins. Repeated 3 times. (f) ROSA26-LSL-spCas9 mice were injected with a pool of AAV to express Cat gRNA in the liver. After 2 weeks, ATGL protein levels were analyzed by indicated antibodies (N = 6). (g) Acoxfl/fl mice were injected with AAV-TBG-Cre virus to ablate hepatic Acox1 (Acox1KO). After 2 weeks, peroxisomal β-oxidation was measured using peroxisomes from livers (N = 5). (h) Hepatic peroxisomes were extracted from the liver specific Acox1 knockout mice to determine cysteine sulfenic acid modification levels of peroxisomal proteins. Repeated 3 times. (i) After harvesting liver from Acox1KO mice, hepatic ATGL levels were checked by IB with indicated antibodies (N = 6). (j) ATGL lipase activity was measured using the liver lysate from Acox1KO mice (N = 6). (k) Atgl transcript in Acox1 knockout liver was quantified by qPCR (N = 6). (l) Peroxisomes were extracted from the livers of mice following acute NAC administration to determine cysteine sulfenic acid modification levels of peroxisomal proteins. Repeated 3 times. (m) NAC was administered acutely by intraperitoneal injection into wild type mice at dosage of 500 mg/kg BW. Hepatic ATGL levels were checked by IB with indicated antibodies (Saline N = 15; NAC N = 17). (n) Quantification of Atgl transcript in the livers of mice following acute NAC administration (Saline N = 11; NAC N = 13). (o) Cysteine sulfenic acid modification in human liver biopsies was analyzed by IB and relative levels of cysteine sulfenic acid modification are presented (50 human samples with different steatosis levels). P = 0.0005 by Spearman test for correlation analysis. (p) Hepatic peroxisomes were extracted from the 14 weeks HFD challenged mice to determine cysteine sulfenic acid modification levels of peroxisomal proteins. Repeated 3 times. (q) After challenging mice with HFD for 16 weeks, hepatic ATGL protein levels were analyzed by indicated antibodies (NCD N = 10; HFD N = 9). (r) Atglfl/fl mice were injected with AAV-TBG-Cre virus to knock out Atgl in liver. After 2 weeks, hepatic peroxisomes were extracted from these mice to determine cysteine sulfenic acid modification levels of peroxisomal proteins. Repeated 3 times. (s) Fatty acid oxidation was measured in liver homogenate from Acox1KO and Pex2KO mice using 14C-labelled palmitate as substrate (N = 6). (t) Ex vivo lipolysis was determined via the measurement of glycerol released from liver pieces dissected from Acox1KO and Pex2KO mice (N = 6). (u) Rates of TG secretion from the livers of Acox1KO and Pex2KO mice were determined by plasma triglyceride accumulation within 4 hours after tail vein injection of tyloxapol (N = 5). (v) Fatty acid uptake rate in the livers of Acox1KO and Pex2KO mice was determined via fluorescence measurement in the liver extracts 30 min after tail vein injection of Bodipy-palmitate at the dose of 10 μg/mouse (N = 5). (w) Fatty acid esterification rate in the livers of Acox1KO and Pex2KO mice was determined via fluorescence measurement in the liver lipid droplet fraction 1 h after tail vein injection of Bodipy-palmitate at the dose of 10 μg/mouse (N = 5). Results are shown as mean ± SEM and analyzed using a Student’s two-sided t test.
