Alternative Mechanism for White Adipose Tissue Lipolysis after Thermal Injury
- 8 Downloads
Extensively burned patients often suffer from sepsis, a complication that enhances postburn hypermetabolism and contributes to increased incidence of multiple organ failure, morbidity and mortality. Despite the clinical importance of burn sepsis, the molecular and cellular mechanisms of such infection-related metabolic derangements and organ dysfunction are still largely unknown. We recently found that upon endoplasmic reticulum (ER) stress, the white adipose tissue (WAT) interacts with the liver via inflammatory and metabolic signals leading to profound hepatic alterations, including hepatocyte apoptosis and hepatic fatty infiltration. We therefore hypothesized that burn plus infection causes an increase in lipolysis of WAT after major burn, partially through induction of ER stress, contributing to hyperlipidemia and profound hepatic lipid infiltration. We used a two-hit rat model of 60% total body surface area scald burn, followed by intraperitoneal (IP) injection of Pseudomonas Aeruginosa-derived lipopolysaccharide (LPS) 3 d postburn. One day later, animals were euthanized and liver and epididymal WAT (EWAT) samples were collected for gene expression, protein analysis and histological study of inflammasome activation, ER stress, apoptosis and lipid metabolism. Our results showed that burn plus LPS profoundly increased lipolysis in WAT associated with significantly increased hepatic lipid infiltration. Burn plus LPS augmented ER stress by upregulating CHOP and activating ATF6, inducing NLRP3 inflammasome activation and leading to increased apoptosis and lipolysis in WAT with a distinct enzymatic mechanism related to inhibition of AMPK signaling. In conclusion, burn sepsis causes profound alterations in WAT and liver that are associated with changes in organ function and structure.
Major burn injury represents one of the most severe forms of trauma and features pervasive stress responses at cellular, tissue and systemic level (1,2). Such stress responses are usually followed by wound infection, systemic inflammatory responses and sepsis that augment the severity of metabolic dysfunction and immunological impairment in the patient (3). Persisting catabolic hypermetabolism, including insulin resistance (IR), hyperglycemia and lipolysis, is the common clinical manifestation in severely burned patients, and these responses are augmented when sepsis is present (4). This can lead to impaired immune function and poor wound healing, and can increase the incidence of multiple organ dysfunction/failure and even death (5,6). We have previously shown in animal models that burn injury plus lipopolysaccharide (LPS) injection induces inflammasome activation in liver, augments hepatic endoplasmic reticulum (ER) stress and liver damage, thus contributing to metabolic derangement (7). Using this model, we observed increased fatty infiltration in liver tissue. This is consistent with the clinical findings that significant hepatic fatty infiltration and hepatomegaly are associated with increased incidence of sepsis and mortality in severely burned patients (8,9); however, the mechanisms underlying the abnormal deposition of lipid in the liver are unclear. Due to the increased appreciation of metabolic cross-talk between liver and WAT in the context of IR and hypermetabolism (10), we hypothesized that increased lipolysis in WAT after severe burn injury and sepsis contributed to hyperlipidemia and hepatic lipid infiltration that led to detrimental outcomes.
The aim of the current study was to determine the underlying mechanisms of lipolysis postburn using a two-hit model of burn plus LPS. We also examined whether hepatic steatosis is linked to pathological changes in WAT.
Materials and Methods
Animal experiments were approved by the Animal Care and Use Committee of Sunnybrook Research Institute in Toronto, Ontario. We follow the Guide for the Care and Use of Laboratory Animals, 2011, adopted by the National Institutes of Health (NIH).
Male Sprague Dawley rats (n = 8 per group), 275 to 300 grams, were purchased from Charles Rivers Laboratory International Inc. and were allowed to acclimate for 1 wk before experiments. Rats were housed in the Sunnybrook Research Institute animal care facility and received a high protein diet (Ensure, #22017C8, Abbott Laboratories) and water ad libitum from arrival until euthanization. Ensure was given from 7 d before the study to adjust the animals to the liquid diet.
The animals were randomized into four groups: sham, burn only, LPS only and burn plus LPS. A well-established method was used to induce a full-thickness scald burn of 60% total body surface area (11,12). Briefly, the animals were anesthetized with IP injection of ketamine (40 mg/kg) and xylazine (5 mg/kg), shaved on both the dorsum and the abdomen and then placed in a mold that exposed a defined area of skin. The exposed skin was lowered into water of 98°C for 10 s on the back and 1.5 s on the abdomen to induce full-thickness scald burn. Lactated Ringer solution (30 ml/kg) was administered IP immediately after the burn for resuscitation. Sham animals were anesthetized and shaved but not burned. The second hit of an IP injection of LPS from Pseudomonas aeruginosa (10 mg/kg, Sigma) was applied 72 h postburn. All the animals were euthanized 24 h after LPS injection (96 h postburn) (7).
3T3-L1 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, 2 mmol/LL glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin in a humidified atmosphere containing 5% CO2 at 37°C. Differentiation of the cells to mature adipocytes was performed as described previously using a differentiation cocktail containing rosiglitazone (13). Adipocytes were serum deprived overnight before the experiment. After the treatment, the cells were briefly washed with phosphate-buffered saline (PBS) and then preserved immediately at −80°C for future analysis.
Plasma and Tissue Collection
Blood was collected into EDTA-containing tubes (30 µL of 0.5 mol/L EDTA). The tubes were placed on ice for at least 30 min and centrifuged at 4°C for 10 min at 1,000g. The plasma supernatant was aliquoted and stored at −80°C for later analysis. Livers were collected after brief portal vein perfusion with PBS (10 mL). Liver and WAT were frozen immediately on dry ice and then stored at −80°C for gene expression and Western blot analyses. Tissue samples were fixed in 10% buffered formalin at 4°C overnight, transferred to 70% ethanol and then paraffin embedded for histology. A second set of tissue samples were embedded in OCT compound (VWR Cat No: 95057-838), frozen on dry ice and stored at −80°C for oil red O (ORO) staining.
Gene Expression Analysis
Total RNA was isolated from liver following manufacturer’s instructions (RNeasy Mini Kit; Qiagen), quantified using a Nanodrop spectrophotometer (NanoDrop Technologies) and reverse transcribed (Applied Biosystems). Real-time quantitative PCR was performed on cDNA with the housekeeping gene rRNA 18S. The sequences of primers of ER stress marker gene CCAAT/-enhancer-binding protein homologous protein (Chop): 5′-AGCGC CTGAC CAGGG AGGTA-3′ and 5′-GCTTG GCACT GGCGT GATGG T-3′.
Antibodies against rat total and phosphorylated AMP-activated protein kinase (AMPKα and phospho-AMPKα), total and phosphorylated protein kinase A catalyst unit (PKA C and phospho-PKA C), hormonal-sensitive lipase (HSL, total and phosphorylated at Ser563, 565, 660 respectively), phosphorylated p44/42 MAPK, perilipin, CHOP, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-7, GAPDH and tubulin were Cell Signaling products. Anti-NOD-like receptor, pyrin domain containing 3 (NLRP3) and anti-ATF6 antibodies were EMD Millipore products. SuperSignal West Pico Chemiluminescent Substrate was a Thermo Scientific Inc. product.
WAT homogenates and whole cell lysates (50 µg of protein per well) were separated by 10% SDS-PAGE gel, proteins were transferred to nitrocellulose membrane as described previously (7) and then blots were probed using the antibodies listed above. Band intensities were detected, normalized and quantified with the Chemidoc and Image Lab 5.0 software (Bio-Rad Laboratories). GAPDH and tubulin were used as loading controls depending on molecular weights of the target proteins.
Immunofluorescent Multichannel Staining of WAT
Antibody staining was performed as described previously (14). Primary antibodies against perilipin, cleaved caspase-3 and CHOP were Cell Signaling products. Fluorophore secondary antibodies (Alexa Fluor 647 Donkey Anti-Mouse and Alexa Fluor 488 Goat Anti-Rabbit) were Life Technologies products. Multiple negative controls of blank, first or second antibodies alone were set up in each batch of experiment to deduct the autofluorescence of the WAT. Mounting media with DAPI was applied for nuclear staining. The percentage of marker-positive cells was determined by taking representative images and directly counting cell numbers. Cell enumerations for each experiment are listed in the text or figure legends.
Hematoxylin and Eosin (H&E), ORO, Immunohistochemical (IHC) and TUNEL Staining of Tissue Sections
H&E and ORO staining of WAT as well as IHC staining of perilipin in liver were performed as described previously (15,16). The size of adipocytes was measured using ImageJ v1.48 (NIH). TUNEL staining of WAT was performed using an immunofluorescent TUNEL staining kit following the manufacturer’s instructions (Promega).
Determination of Free Fatty Acid (FFA), Glycerol and Triglyceride Levels in Blood
Levels of FFA, glycerol and triglyceride in the blood were determined using FFA, glycerol and triglyceride colorimetric assay kits according to the manufacturer’s instructions (Cayman Chemical).
The statistical analysis was performed using Prism version 5.01 (GraphPad Software). P < 0.05 was considered statistically significant.
All supplementary materials are available online at https://doi.org/www.molmed.org .
Burn and LPS Induce Significant Catabolism and Hepatic Fatty Infiltration
Increased WAT Lipolysis in the Two-Hit Rat Model of Burn plus LPS
Increased Lipolysis in WAT after Burn plus LPS Is Associated with Reduced AMPK Signaling
Burn plus LPS Increases Adipocyte Apoptosis
Burn and LPS Synergistically Induce Apoptosis in WAT
Increased Macrophage Infiltration and Inflammasome Activation Correlated with Apoptosis in WAT
Using the rat two-hit model of severe burn injury followed by LPS IP injection, we mimicked the clinical scenario of burn followed by sepsis and investigated the lipid metabolism cross-talk between WAT and liver during critical illness. We found that there is significant more WAT lipolysis and hepatic fatty infiltration in burn plus LPS compared with burn alone. Mechanistically, we attribute the enhanced lipolysis in WAT mainly to the degradation of the perilipin coating of the lipid droplet and to the impairment of AMPK signaling as well as its downstream lipolysis-inhibiting HSL phosphorylation at Ser565. We also found that augmented ER stress, NLRP3 inflammasome activation and apoptosis converged to enhance WAT lipolysis under conditions of severe acute stress.
WAT and liver are in constant communication with one another. For example, JNK1 activation in WAT causes diet-induced hepatic IR (20). Moreover, inhibition of hepatic eIF2α (involved in protein synthesis and ER stress) impairs WAT insulin sensitivity (21). In the current study, we demonstrated that increased WAT apoptosis is positively correlated with lipid infiltration in the liver, implicating increased WAT lipolysis as a source of lipid for deposition in the liver. While this partly explains the morphologic changes in the liver after severe burn, further investigation is needed to determine the mechanisms underlying pronounced hepatic steatosis following thermal injury. It is also possible that decreased fatty acid oxidation and outbound lipid transportation due to impaired hepatocyte function contribute to increased fat content in the liver.
Interestingly, we did not observe a significant elevation of circulating FFA, glycerol or triglycerides in burn, LPS or burn plus LPS groups (Supplementary Figure 4). There are two possible explanations for this. First, plasma lipid concentration represents an equilibrium between what is released and what is taken up and thus may not reflect the changes in flux (22), as is indicated in high fat-fed mice which exhibit no significant increases in blood FFA or glycerol (23). Secondly, considering the severity of the intervention of burn plus LPS, we did not fast the animals before collecting the blood samples. Differences in food intake of the animals would significantly affect the plasma FFA, glycerol and triglyceride levels. Nonetheless, the correlation of TUNEL staining in WAT and ORO staining in liver suggests that increased lipolysis in WAT contributes to the lipid infiltration in the liver. Hepatic steatosis contributes to augmented hepatic ER stress, mitochondrial dysfunction and insulin resistance (24,25). Moreover, it may impair the hepatic clearance of LPS (26). As shown in the current animal study and in other clinical observations (17,27), LPS strongly activates lipolysis in WAT, which could instigate a vicious positive feedback loop between WAT lipolysis, hepatic lipid accumulation and hepatic LPS clearance.
As a mechanism of energy reservation and buffering, lipolysis of WAT is regulated by multiple signaling pathways (28,29). The first step of lipid mobilization from WAT is the phosphorylation or degradation of perilipin and activation of desnutrin/adipose triglyceride lipase (ATGL), which converts triacylglycerol (TAG) to diacylglycerol (DAG). Activated HSL then converts DAG into 2-monoacylglycerol (MAG), which, in turn, is broken down by monoacylglycerol lipase (MGL) into FFA and glycerol. HSL can convert TAG to DAG and has long been considered the key regulator of lipolysis and its activation the driving force for hyperlipidemia since MGL is abundant and its catalyzing activity is not rate-limited (30). This is true in low-grade, chronic adipose stress conditions such as obesity and diabetes, where increased lipolysis occurs in parallel to activation of PKA and HSL phosphorylation at Ser563, Ser660 (31). Based on our observations, HSL and its upstream modulators PKA and MAPK were all suppressed under severe and acute traumatic stress conditions, yet we still observed augmented lipolysis and even more severe ectopic lipid deposition in liver. In the current study, we demonstrated that the inhibition of AMPK and its downstream phosphorylation of HSL at Ser565 contribute to activation of lipolysis in WAT under severe stress conditions.
We also observed increased apoptosis in WAT, which has been implicated in the enhancement of lipolysis (32). To address the causative factors of increased apoptosis, we first determined whether there was augmented ER stress in WAT after burn plus LPS since it is well accepted that ER stress triggers apoptosis under multiple circumstances (32,33). Here, we showed that burn injury alone significantly induces ER stress in WAT. Together with LPS-mediated proapoptotic signaling, burn plus LPS further stimulated ER stress and apoptosis, as shown by the robust activation of apoptosis-related ER stress markers CHOP and ATF6 as well as enhanced TUNEL staining. Changes in the proapoptotic signaling indicated that ER stress induced by burn injury mainly activated the intrinsic pathway of apoptosis that is manifested by activation of caspase-9 and increased cleavage of caspase-3 (34). Caspase-7 is a direct substrate of caspase-1, one of the products of inflammasome activation. As such, significantly increased cleavage of caspase-7 in LPS and burn plus LPS groups suggests the contribution of inflammation and inflammasome activation on apoptosis (35).
We have previously reported the activation of the inflammasome in the WAT of burn patients (36). As the WAT was collected from the wound or adjacent area in these experiments, this raises the question whether inflammasome activation also occurs in distal tissue and organs. In the current study, we observed in experimental animals that severe burn injury and LPS trigger macrophage infiltration and subsequent NLRP3 inflammasome activation in WAT distal to burn wound. It has long been postulated that the danger-associated molecular pattern molecules (DAMPs) contribute to inflammasome activation (37,38). While the identity of the DAMP(s) involved is undetermined to date, augmented ER stress may be responsible for the production of DAMPs considering the concomitance of ER stress and inflammasome activation in WAT (39). Furthermore, since the outcome of NLRP3 inflammasome activation is the maturation of proinflammatory cytokine IL-1β, which then contributes to increased lipolysis, IR and hyperglycemia, the resulting prolonged hyperglycemic response further enhances and sustains inflammasome activation and proinflammatory responses, serving as another positive feedback loop contributing to increased postburn morbidity and mortality (40,41).
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This research was supported by the National Institutes of Health (R01-GM087285-01), Canadian Institutes of Health Research (123336), the CFI Leaders Opportunity Fund (25407) and the Health Research Grant Program. We thank Cassandra Belo for her technical assistance and proofreading. We thank Abdikarim Abdullahi for his assistance in animal experiments. We are grateful to Sheila Costford for her final proofreading and editing of the manuscript.
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.
The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
To view a copy of this license, visit (https://doi.org/creativecommons.org/licenses/by-nc-nd/4.0/)