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, 10 (12), 870

Browning of White Adipose Tissue After a Burn Injury Promotes Hepatic Steatosis and Dysfunction

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Browning of White Adipose Tissue After a Burn Injury Promotes Hepatic Steatosis and Dysfunction

Abdikarim Abdullahi et al. Cell Death Dis.

Abstract

Burn patients experiencing hypermetabolism develop hepatic steatosis, which is associated with liver failure and poor outcomes after the injury. These same patients also undergo white adipose tissue (WAT) browning, which has been implicated in mediating post-burn cachexia and sustained hypermetabolism. Despite the clinical presentation of hepatic steatosis and WAT browning in burns, whether or not these two pathological responses are linked remains poorly understood. Here, we show that the burn-induced WAT browning and its associated increased lipolysis leads to the accelerated development of hepatic steatosis in mice. Deletion of interleukin 6 (IL-6) and the uncoupling protein 1 (UCP1), regulators of burn-induced WAT browning completely protected mice from hepatic steatosis after the injury. Treatment of post-burn mice with propranolol or IL-6 receptor blocker attenuated burn-induced WAT browning and its associated hepatic steatosis pathology. Lipidomic profiling in the plasma of post-burn mice and burn patients revealed elevated levels of damage-inducing lipids (palmitic and stearic acids), which induced hepatic endoplasmic reticulum (ER) stress and compromised hepatic fat oxidation. Mechanistically, we show that hepatic ER stress after a burn injury leads to a greater ER-mitochondria interaction, hepatocyte apoptosis, oxidative stress, and impaired fat oxidation. Collectively, our findings uncover an adverse "cross-talk" between the adipose and liver tissue in the context of burn injury, which is critically mediated by WAT browning.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Browning of white adipose tissue leads to the development of hepatic steatosis post-burn injury.
a, b Changes in total body (a) and adipose tissue (b) weight in post-burn and control mice. c Analysis of oxygen consumption rate in isolated inguinal WAT of post burned mice and controls. d Plasma concentration of free fatty acids in burned mice and controls. e Representative macroscopic pictures of isolated inguinal WAT from burned mice and controls at autopsy. f Quantitative RT-PCR analysis of browning gene Uncoupling protein 1 (UCP1) in inguinal WAT of burned mice and controls. g Immunoblot analysis of UCP1 in inguinal WAT of burned mice and controls. h H&E and UCP1 staining in inguinal WAT of burned mice and controls. i Liver weights normalized to body weight of burned mice and controls. j Oil Red O staining for fat droplets in liver sections from burned mice and controls. k Triglyceride (TG) content of livers from burned mice and controls. l Quantitative RT-PCR analysis of lipogenic genes was measured in livers from burned mice and controls. m Immunoblot analysis of lipogenic proteins in livers from burned mice and controls. Data represented as mean ± SEM, p < 0.05 *significant difference burn vs. controls (n = 8, biological replicates, experiments repeated two times).
Fig. 2
Fig. 2. IL-6−/− and UCP-1−/− KO mice are protected from burn-induced browning and hepatic steatosis post-injury.
a, b Changes in total body (a) and fat (b) weight in IL-6−/− burned mice and IL-6−/− controls. c Plasma concentration of free fatty acids in IL-6−/− burned mice and controls. d UCP1 staining in inguinal WAT of WT and IL-6−/− burned mice and controls. e Quantitative RT-PCR analysis of browning gene UCP1 in inguinal WAT of wild type (WT) and IL-6−/− burned mice and controls. f Liver weights normalized to body weight of WT and IL-6−/− burned mice and controls. g Oil Red O staining for fat droplets in liver sections from WT and IL-6−/− burned mice and controls. h Triglyceride (TG) content of livers from WT and IL-6−/− burned mice and controls. i H&E and UCP1 staining in inguinal WAT of WT and UCP-1−/− burned mice and controls. j Quantitative RT-PCR analysis of browning gene UCP1 in inguinal WAT of WT and UCP-1−/− burned mice and controls. k Plasma concentration of free fatty acids in UCP-1−/− burned mice and controls. l Oil Red O staining for fat droplets in liver sections from WT and UCP-1−/− burned mice and controls. m Triglyceride (TG) content of livers from WT and UCP-1−/− burned mice and controls. Data represented as mean ± SEM, p < 0.05 *significant difference WT burn vs. controls, p < 0.05 # WT burn vs. IL-6−/− / UCP-1−/− (n = 7, biological replicates, experiments repeated two times).
Fig. 3
Fig. 3. Blockage of IL-6 signaling post burn injury attenuates burn-induced WAT browning and hepatic steatosis.
a, b Changes in total body (a) and fat (b) weight in post burn mice treated with vehicle or anti-mouse IL-6R monoclonal antibody daily for 5 days. c Mitochondrial coupling efficiency in inguinal WAT isolated from post burn mice treated with vehicle or anti-mouse IL-6R monoclonal antibody. d H&E and uncoupling protein 1 (UCP1) staining in inguinal WAT of post burn mice treated with vehicle or anti-mouse IL-6R monoclonal antibody. e Quantitative RT-PCR analysis of browning genes in inguinal WAT of post burn mice treated with vehicle or anti-mouse IL-6R monoclonal antibody. f Liver weights normalized to body weight of post burn mice treated with vehicle or anti-mouse IL-6R monoclonal antibody. g Oil Red O staining for fat droplets in liver sections from post burn mice treated with vehicle or anti-mouse IL-6R monoclonal antibody. h Triglyceride (TG) content of livers from post burn mice treated vehicle or anti-mouse IL-6R monoclonal antibody. Data represented as mean ± SEM, p < 0.05 *significant difference WT burn vs. littermate controls, p < 0.05 # WT burn vs. IL-6−/− burn (n = 7, biological replicates, experiments repeated two times).
Fig. 4
Fig. 4. Lipodomic profiling after a burn injury reveals upregulation of ER stress inducing lipids.
a, b Heat map display of lipid species within the plasma taken from burn patients and post-burn mice. c Quantification of FFAs (Palimitic and Stearic) in the plasma of wild type burned mice and controls. d Immunoblot of ER stress marker p-eif2a in HepG2 cells exposed to vehicle or Palimitate (100 μm or 500 m) for 24 h. e Oxygen Consumption Rate (OCR) in HepG2 cells exposed to vehicle or Palimitate (500μm) for 24 h. f Quantitative RT-PCR analysis of ER stress/UPR gene expression from livers of wild type (WT) burned mice and controls. g Immunoblot analysis of ER stress/UPR proteins in liver samples of wild type (WT) burned mice and controls. h, i Quantitative RT-PCR analysis ER stress/UPR and CPEB4 gene expression from the livers of UCP-1−/− and IL-6−/− burned mice and controls. j Parametric analysis of gene-set enrichment (PAGE) of the most highly upregulated (red) and down-regulated (blue) mitochondrial genes in livers of wild type (WT) burned mice and controls. k Quantitative RT-PCR analysis of beta-oxidation genes in the livers of wild type (WT) burned mice and controls. Data represented as mean ± SEM, p < 0.05 *significant difference WT burn vs. controls (n = 8, biological replicates, experiments repeated two times).
Fig. 5
Fig. 5. Hepatic ER stress post-burn injury leads to increased ER-Mitochondrial interaction.
a, b Representative TEM images and quantification (scale bare = 100 nm) illustrating ER-Mitochondria tethering in liver sections derived from wild type (WT) burned mice and controls. c, d Representative PLA images (×63 magnification) and quantification of VDAC1/IP3R1 interactions (small red dots around the nucleus) in livers from wild type (WT) burned mice and controls. e Representative western blot and quantification of MAM enriched protein IP3R1 in livers of wild type (WT) burned mice and controls. f Representative TEM images (scale bare = 100 nm) illustrating ER-mitochondria tethering of liver sections derived Sham, Burn, and Tun + Burn mice. g Representative western blot of MAM enriched protein IP3R1 in livers of Sham, Burn, Tun., and Tun + Burn mice. h, i Representative PLA images (×63 magnification) and quantification of VDAC1/IP3R1 interactions (small red dots around the nucleus) in livers of Sham, Burn, Tun., and Tun + Burn mice. j Parametric analysis of gene-set enrichment (PAGE) of the most highly upregulated (red) and down-regulated (blue) mitochondrial genes in livers of burned mice and Tun + Burn mice. Data represented as mean ± SEM, p < 0.05 *significant difference WT burn vs. control, p < 0.05 # WT burn vs. Tun + Burn (n = 8, biological replicates, experiments repeated two times).
Fig. 6
Fig. 6. A cell permeant IP3R peptide attenuates ER stress and blocks apoptosis in vitro.
a A schematic illustrating the consequences of increased hepatic ER stress and MAM enrichment that can lead to an efflux of excessive Ca2+ from the ER via the IP3R receptor to the mitochondria. The by-product of mitochondrial damage (cytochrome c) feedbacks to the IP3R to sustain this cycle that ultimately results in cellular apoptosis. b Immunoblot of ER stress proteins c-ATF6 and p-jnk in HepG2 cells treated with either TG (100 nM) for 24 h or vehicle. c Immunofluorescence staining of ER stress marker BiP (red) in HepG2 cells treated with either TG (100 nM) for 24 h or vehicle. d Immunoblot and quantification of ER stress protein p-eif2α from HepG2 cells treated with either TG (100 nM) for 24 h, TG (100 nM) treated cells incubated with either BODIPY-IP3RCYT-MUT(400 nM), and or BODIPY-IP3R-CYT(400 nM) for 4 h. e Immunofluorescence staining of pro-apoptotic marker CHOP (green) in HepG2 cells treated with either TG (100 nM) for 24 h, TG (100 nM) treated cells incubated with either BODIPY-IP3RCYT-MUT(400 nM), and or BODIPY-IP3RCYT(400 nM) for 4 h. f Quantification of Immunofluorescence staining of CHOP from (e). g Immunofluorescence staining of mitochondria with Deep Red MitoTracker (red) in HepG2 cells treated with either TG (100 nM) for 24 h, TG (100 nM) treated cells incubated with either BODIPY-IP3RCYT-MUT(400 nM), and or BODIPY-IP3RCYT(400 nM) for 4 h. h Quantification of Immunofluorescence staining of the MitoTracker from (g). Data represented as mean ± SEM, *p < 0.05 vs. control or TG, ϕp < 0.05 vs. control #p < 0.05 vs. IP3RCYT-MUT/ TG (n = 7, biological replicates, experiments repeated two times). Scale bars represent ×20 magnification for (c, e), and ×60 magnification for (g).
Fig. 7
Fig. 7. Schematic diagram illustrating the consequences of WAT browning and its associated lipotoxicity for the liver after a burn injury.
Following a burn injury you have the activation of the browning process whereby the white adipose tissue converts to beige fat. Pro-inflammatory FFAs released from beige fat than travel to the liver causing hepatic steatosis and hepatic dysfunction by activating the ER stress response in hepatocytes. Hepatic ER stress then leads to increased expression of the MAM enriched protein IP3R1, which leads to a greater ER-Mitochondria interaction and facilitates Ca2+ transfer from ER (via IP3R1) to the mitochondria. This, in turn, can lead to reductions in mitochondrial fat oxidation. If this chronic ER stress mediated by lipid infiltration is not mitigated it can ultimately result in cellular apoptosis.

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