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Sepsis Induces Adipose Tissue Browning in Nonobese Mice But Not in Obese Mice

Ayalon, Itay*; Shen, Hui*; Williamson, Lauren*; Stringer, Keith; Zingarelli, Basilia*; Kaplan, Jennifer M.*

doi: 10.1097/SHK.0000000000001076
Basic Science Aspects
Editor's Choice

ABSTRACT Severe sepsis and septic shock are the biggest cause of mortality in critically ill patients. Obesity today is one of the world's greatest health challenges. Little is known about the extent of involvement of the white adipose tissue (WAT) in sepsis and how it is being modified by obesity. We sought to explore the involvement of the WAT in sepsis. We hypothesize that sepsis induces browning of the WAT and that obesity alters the response of WAT to sepsis. Six-week-old C57BL/6 mice were randomized to a high-fat diet to induce obesity (obese group) or control diet (nonobese group). After 6 to 11 weeks of feeding, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Mice were sacrificed at 0, 18, and 72 h after CLP and epididymal WAT (eWAT), inguinal WAT, and brown adipose tissue (BAT) harvested. Both types of WAT were processed for light microscopy and transmission electron microscopy to assess for morphological changes in both obese and nonobese mice. Tissues were processed for immunohistochemistry, image analyses, and molecular analyses. BATs were used as a positive control. Nonobese mice have an extensive breakdown of the unilocular lipid droplet and smaller adipocytes in WAT compared with obese mice after sepsis. Neutrophil infiltration increases in eWAT in nonobese mice after sepsis but not in obese mice. Nonobese septic mice have an increase in mitochondrial density compared with obese septic mice. Furthermore, nonobese septic mice have an increase in uncoupling protein-1 expression. Although the WAT of nonobese mice have multiple changes characteristic of browning during sepsis, these changes are markedly blunted in obesity.

*Division of Critical Care Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio

Division of Pathology and Laboratory Medicine, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio

Address reprint requests to Jennifer M. Kaplan, MD, MS, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, MLC 2005, Cincinnati, OH 45229. E-mail: Jennifer.Kaplan@cchmc.org

Received 10 August, 2017

Revised 6 September, 2017

Accepted 30 November, 2017

NIH R01 GM126551 (JMK), R01 GM067202 (BZ), and P30DK078392.

The authors report no conflicts of interest.

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INTRODUCTION

Sepsis is a syndrome of physiologic, pathologic, and biochemical abnormalities induced by infection. The clinical definition of sepsis in adults was revised based on the current understanding of sepsis-induced changes in organ and tissue function (1). Severe sepsis and septic shock are the biggest causes of mortality in critically ill patients (2). A recent Centers for Disease Control and Prevention (CDC) report suggests that as many as 6% of all reported deaths in the United States could be attributed to sepsis (3).

Obesity is a global epidemic and is one of the world's greatest health challenges, contributing to chronic diseases and burdening health services (4). Morbid obesity is independently associated with increased sepsis risk (5). The effects of obesity on mortality remain controversial as overweight and obese patients may have protection from critical illness in certain diseases, termed the “obesity paradox.” Recent studies in adults with obesity and critical illness demonstrated lower mortality rates than nonobese patients (6, 7), which is in contrast to early data suggesting the opposite (8). In the pediatric population the data are scant, but there is some evidence suggesting positive association between increased body mass index and mortality (9, 10).

Sepsis induces multisystem organ dysfunction in many organs, including the heart, lungs, and kidney. These organs are extensively studied both in animal models and translational clinical studies. The adipose tissue, however, remains a neglected tissue. No longer considered as simply a storage organ for lipids, the adipose tissue is a metabolically dynamic organ capable of synthesizing several biologically active compounds that regulate metabolic homeostasis. As sepsis is a state in which the metabolic homeostasis is heavily deranged, it is only logical to assume that adipose tissue is involved in the process of sepsis, like any other major organ and therefore deserves more attention.

Recent evidence suggests that adipose tissue undergoes a transformation process known as “browning” in which white adipose tissue (WAT) can adopt a brown adipose tissue (BAT) phenotype in response to various stimuli (11). One of the characteristics of browning is the expression of the uncoupling protein (UCP)-1 (12). UCP1 is a small protein located on the inner membrane of the mitochondria, facilitating proton transport and dissipating the proton gradient while allowing mitochondrial membrane potential to be transduced to heat in a nonshivering pathway (thermogenesis) (13). The process of browning was previously described in states characterized by increased adrenergic stimuli such as cold exposure (14), chronic exercise (15), and burns (16). Sepsis is another state characterized by increased adrenergic stimuli, but little is known about the association between sepsis and browning. We hypothesize that sepsis induces browning of the WAT, but obesity alters this adipose tissue response during sepsis.

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MATERIALS AND METHODS

Animals

The investigations conformed to the Guide for the Care and Use of Laboratory Animals (17) and were approved by the Institutional Animal Care and Use Committee at Cincinnati Children's Hospital Medical Center. Male C57BL/6 mice at 6 weeks of age were obtained from Charles River Laboratories International, Inc. (Wilmington, Mass). The mice were housed in the animal facility at the Cincinnati Children's Research Foundation. Food and water were provided ad libitum.

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Dietary intervention

At the age of 6 weeks, animals were randomized to a high-fat diet (TestDiet—58Y1) (60% kcal provided by fat) or a standard control diet (Formulab—5008) (16% kcal provided by fat) for 6 or 11 weeks as indicated. Given the small amount of adipose tissue obtained from nonobese mice, it was necessary to extend the dietary intervention beyond 6 weeks for some experiments. Every high-fat feeding group had its own age-matched control group that received the same duration of dietary intervention. Body weights were monitored weekly throughout the diet phase and are demonstrated in Figure 1 by age cohort at the time of cecal ligation and puncture (CLP).

Fig. 1

Fig. 1

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Induction of polymicrobial sepsis by CLP

After 6 to 11 weeks of dietary intervention (at 12 or 17 weeks of age), polymicrobial sepsis was induced by CLP as previously described (18–20). Animals were anesthetized with isoflurane. After opening the abdomen, the cecum was exteriorized and ligated by a 6.0 silk ligature at its base without obstructing intestinal continuity. The cecum was punctured twice with a 22-gauge needle and returned to the peritoneal cavity. The abdominal incision was closed with silk running sutures and liquid topical adhesive. After the procedure, animals were fluid resuscitated with sterile saline (1 mL) injected subcutaneously. All animals received a single dose of intraperitoneal antibiotic (Imipenem 25 mg/kg) and a single dose of subcutaneous analgesia (Buprenorphine 0.05 mg/kg). Animals within each dietary cohort were randomized to either the control group (no CLP—0 h) or were sacrificed at 18 or 72 h after CLP (n = 4–6 animals/group). Plasma, epididymal WAT (eWAT), inguinal WAT (iWAT), and intrascapular BAT were collected for analysis. BATs were used as a positive control.

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Immunohistochemistry

Harvested samples of eWAT, iWAT, and BAT were fixed in formalin and embedded in paraffin. Sections were stained using hematoxylin and eosin (H&E) and automated immunohistochemistry (IHC) was performed (Ventana Medical Systems, Tucson, Ariz) using anti-UCP1 and anti-myeloperoxidase (MPO) antibodies (Abcam, Cambridge, Mass). Whole slide digital images were obtained from the resultant slides using an Aperio AT2 digital slide scanner (Leica Microsystems, Buffalo Grove, Ill). The high-resolution digital images were analyzed using Image Scope software (Aperio version 12) using the positive pixel count algorithm as described by Gannon et al. (21). In brief, the algorithm generates three classes of output values (weak positive, positive, and strong positive) based on pixel intensity that represents staining intensity. Only positive and strong positive values were included in the analysis. Three similar-sized areas were randomly selected on each digital slide, and the average value of those three areas was used as the final value for comparison.

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Adipose lipid droplet size measurement

Whole slide digital images of mice at 17 weeks of age were obtained for each specimen as described above for IHC. Each image was analyzed using ImageJ software (version 1.48) (22). In brief, three areas in each slide were randomly selected. Twenty adjacent adipocytes in each area were selected and measured for the total lipid droplet surface area. The average lipid droplet surface area was calculated for each sample. The frequency of each adipocyte size was counted.

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Transmission electron microscopy

Fresh eWAT, iWAT, and BAT were fixed in 2.5% glutaraldehyde and 1% paraformaldehyde. Samples were postfixed in 1% osmium tetroxide in 0.15 M sodium cacodylate buffer, processed through a graded series of alcohols, infiltrated, and embedded in LX-112 resin. After polymerization at 60° for 3 days, ultrathin sections (120 nm) were cut using a Leica EM UC6 ultramicrotome (Leica Microsystems, Buffalo Grove, Ill) and counterstained in 2% aqueous uranyl acetate and Reynold's lead citrate. The ultrathin sections were examined with a Hitachi H-7650 transmission electron microscopy (TEM) equipped with an AMT digital camera using the AMT Capture Engine 2 software.

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Mitochondrial density

Digital TEM images in a predefined magnification (×3,000) were used to count the number of mitochondria seen adjacent to the nucleus. To compare the number of mitochondria in each sample, mitochondria number was adjusted to the total surface area in which the mitochondria were located, and termed the “mitochondrial density.”

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Western blot analysis

Mitochondria were extracted and isolated from frozen eWAT and iWAT (∼300 mg) using mitochondria extraction and isolation kits after the manufacturer's protocol (Miltenyi Biotec, Auburn, Calif). The Invitrogen NuPAGE gel electrophoresis system (Invitrogen) was used for Western blotting. NuPAGE 10% Bis-Tris gels were used with NuPAGE MOPS buffer and Invitrogen Novex Mini-Cell, BioRad PowerPac 300. Membranes imaged using BioRad ChemiDoc XRS+ gel documentation system and analyzed using ImageLab (version 5.1) software (BioRad, Hercules, Calif). The following antibodies were used: anti-UCP1 (Sigma-Aldrich, St. Louis, Mo), mitochondrial antivoltage-dependent anion-selective channel (VDAC)-1 protein (Santa Cruz Biotechnology, Dallas, Tex).

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Enzyme-linked immunosorbent assay

Mitochondria were extracted and isolated from frozen eWAT and iWAT in the same manner as described above in mice aged 17 weeks. UCP1 levels were measured using a mitochondrial UCP1 enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's protocol (MyBioSource, San Diego, Calif). Light absorbance was measured at 450 nm using a spectrophotometer (Spectramax plus 384, Molecular Devices). The data were normalized to the original tissue weight and expressed as picogram of UCP1 per gram of tissue.

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Measurement of nonesterified fatty acids and triglycerides

Plasma samples from mice aged 17 weeks were measured by enzymatic kit for nonesterified fatty acids (Wako Diagnostics, Mountain View, Calif) and triglycerides (Caymen Chemicals, Ann Arbor, Mich) per the manufacture's protocol.

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Statistical analysis

Data were analyzed using SigmaPlot for Windows Version 13 (SysStat Software, San Jose, Calif). For data comparison among two or more interventions, statistical analysis was performed using the two-way ANOVA with Holm–Sidak method for parametric data and the Kruskal–Wallis ANOVA with the Dunn post hoc test for nonparametric data. Data are expressed as mean and SD for parametric data in the text and figures. A value of P ≤ 0.05 was considered significant.

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RESULTS

Sepsis induces WAT remodeling in nonobese mice that is impaired in obese mice

WAT is a dynamic and modifiable tissue. With the development of obesity, WAT undergoes a process of tissue remodeling in which adipocytes increase in both number (hyperplasia) and size (hypertrophy). We sought to determine the differences in adipocyte size and determine the changes in adipose tissue in obese and nonobese 12-week-old mice during sepsis. We used H&E stained eWAT and iWAT slides to examine morphologic differences between obese and nonobese mice during sepsis (Fig. 2). As expected in eWAT before sepsis (CLP 0 h), nonobese mice have smaller adipocytes than obese mice (Fig. 2—e1, e3 vs. e2, e4). At 18 and 72 h after CLP, adipocytes decrease in size in nonobese mice and there is an increase in cellular infiltration when compared with before sepsis (Fig. 2—e5, e7, e9, e11). However, in obese mice, adipocyte size does not change during sepsis when compared with before sepsis and there is not a profound cellular infiltration (Fig. 2—e6, e8, e10, e12).

Fig. 2

Fig. 2

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Sepsis causes a significant reduction in lipid droplet surface area in nonobese mice but to a lesser extent in obese mice

To quantify adipocyte size, we measured the mean lipid droplet area in eWAT and iWAT from 17-week-old obese and nonobese mice at 0 and 18 h after CLP. The mean lipid droplet area in eWAT was lower in nonobese mice compared with obese mice at baseline (0 h CLP = nonseptic) (3,371 ± 1,841 μm2 vs. 8,606 ± 1,234 μm2, P < 0.001) (Fig. 3, A and C). After sepsis, a 53% reduction in droplet size occurred in nonobese mice compared with baseline (1,564 ± 511 μm2, P ≤ 0.05). Septic obese mice had only an 18% reduction in droplet size after sepsis and remained significantly higher than nonobese septic mice (7,007 ± 647 μm2, P ≤ 0.05). Interestingly, in general, nonobese mice showed more homogeneity in lipid droplet size than obese mice regardless of sepsis state. Similar droplet size changes were found in iWAT (Fig. 3, B and C).

Fig. 3

Fig. 3

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Neutrophil infiltration to the eWAT is increased during sepsis in nonobese mice but not in obese mice

To immunohistochemically verify that the cellular infiltrate demonstrated in eWAT after CLP (Fig. 2—e3, e7) was composed of neutrophils, we used an antibody against the neutrophil marker, MPO, and then quantified the positive pixel count using image analysis software. EWAT from nonobese mice showed an increase in MPO positive pixel count after sepsis compared with nonobese mice at baseline (6,820 ± 4,111 vs. 1,569 ± 335 positive pixel count, P < 0.01) and compared with obese mice after sepsis (2,310 ± 1,233 positive pixel count, P = 0.001). Surprisingly, MPO positivity was low in iWAT and did not increase after sepsis in obese or nonobese mice.

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Sepsis induces a breakdown of the unilocular lipid droplet in nonobese mice but not in obese mice

To further investigate the alterations in WAT morphology during sepsis, we examined tissue using TEM in mice aged 12 weeks. In nonobese mice, we found an extensive breakdown of the characteristic unilocular lipid droplet into many different sized lipid droplets (lipid breakdown). Smaller lipid droplets formed from a larger droplet in a process resembling “budding” in which the smaller lipid seemed to be pinched off from the larger droplet (Fig. 4, A and B). Budding was evident at 18 h after CLP, but was more extensive at 72 h after CLP (Fig. 5—e9, e11, i9, and i11). Budding was abundant in nonobese mice, but could only be seen sporadically and to a lesser degree in obese mice (Fig. 5). In many instances, these newly formed lipid droplets are near mitochondria with direct physical contact between the outer layers of the lipid droplet and mitochondria (Fig. 4E).

Fig. 4

Fig. 4

Fig. 5

Fig. 5

The morphological changes highly suggest that lipolysis is occurring in these tissues. To determine if fatty acids are released during this process, we measured fatty acid levels in mice aged 17 weeks. There was no difference in fatty acid levels between nonobese and obese mice at baseline (1.8 ± 0.54 mmol/L and 1.7 ± 0.5, respectively). However, fatty acid levels decreased in both nonobese and obese mice after sepsis (1 ± 0.2 mmol/L and 0.96 ± 0.2, respectively), but there was no dietary effect. We also found no difference in plasma triglyceride levels between nonobese and obese mice at baseline (80 ± 15 mg/dL and 88 ± 23, respectively). Like fatty acid levels, there was a significant decrease in triglyceride levels after sepsis in nonobese and obese mice compared with baseline (39 ± 10 mg/dL and 56 ± 11, respectively). However, triglyceride levels were significantly lower in nonobese septic mice than obese septic mice (P ≤ 0.05).

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Mitochondrial density increases during sepsis in nonobese mice but not in obese mice

While scanning WAT for ultrastructural changes using TEM, we noticed an increase in the number of mitochondria in the nonobese septic group (Fig. 5—e7 and e11) when compared with before sepsis (Fig. 5—e3) and compared with the obese septic group (Fig. 5—e8, e12). To quantify the increase in mitochondrial load, we counted the mitochondria in a predefined magnification and adjusted it to the measured surface area to determine the mitochondrial density. Mitochondrial density was significantly higher at 18 h after CLP in nonobese mice than nonseptic mice in both eWAT and iWAT (Fig. 6). Mitochondrial density did not change at 18 h after CLP in obese mice and was significantly lower than nonobese mice (Fig. 6).

Fig. 6

Fig. 6

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UCP1 expression increases in nonobese mice after sepsis but not in obese mice

Many of the changes described above (i.e., multiple small lipid droplets, increased extracellular matrix between lipid droplets and numerous mitochondria) are known characteristics of BAT and the browning process. To provide further evidence of browning during sepsis, we investigated changes in the key marker of browning, UCP1. As UCP1 is a mitochondrial protein, we extracted and isolated eWAT mitochondria and evaluated the mitochondrial extract for UCP1 expression using Western blot analysis. As demonstrated in Figure 7A, UCP1 expression increased in nonobese mice after sepsis (18 h after CLP), but did not increase in obese mice after sepsis. As further evidence of changes in UCP1 expression, we measured UCP1 expression in isolated mitochondrial extracts from eWAT and iWAT by ELISA. As demonstrated in Figure 7B, UCP1 expression was higher in eWAT from nonobese septic mice than nonobese nonseptic mice (369 ± 138 vs. 223 ± 72 pg/gr tissue, P < 0.05) and than septic obese mice (226 ± 114 pg/gr tissue, P < 0.01). Similar changes were demonstrated in iWAT (Fig. 7C).

Fig. 7

Fig. 7

To confirm the above findings, we used IHC to determine UCP1 expression and quantified the positive pixel count using image analysis software (ImageScope), eWAT. UCP1 expression was higher at 18 h after CLP in nonobese mice after sepsis than baseline (CLP 0 h) (Fig. 8A—e3, e1; B). Obese septic mice had no significant change in UCP1 expression compared with baseline and expression remained significantly lower than nonobese septic mice (Fig. 8A—e4, e2; B). Similar findings were demonstrated in iWAT (Fig. 8, A and C).

Fig. 8

Fig. 8

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DISCUSSION

In this study, we demonstrate that sepsis induces adipose tissue browning in nonobese mice but not in obese mice. We found that sepsis induces a breakdown of the WAT unilocular lipid droplet. This leads to a significant reduction in lipid droplet surface area in nonobese mice and to a lesser extent in obese mice during sepsis. Mitochondrial density and UCP1 expression increase during sepsis in nonobese mice but not in obese mice. These morphological changes in nonobese mice are consistent with WAT browning.

Our data demonstrate that during sepsis lipid droplets decrease in size and the unilocular lipid droplet breaks down into multiple smaller droplets, a process called “budding.” This occurs in nonobese mice more than in obese mice. Langouche et al. demonstrated that critically ill patients had smaller adipocytes than healthy controls (23). We found similar changes in our murine model of sepsis in which nonobese mice had ∼50% decrease in the lipid droplet size during sepsis. Although obese mice had a statistically significant decrease in adipocyte size, this only represented an 18% reduction in adipocyte size during sepsis. Smaller sized adipocytes may represent newly formed adipocytes or cellular breakdown of larger adipocytes. Our TEM results in nonobese septic mice demonstrating budding of lipid droplets provide support that smaller adipocytes represent cellular breakdown and lipolysis.

Lipolysis is one of the main functions of adipose tissue and during this process free fatty acids are released for energy utilization. Lipolysis is a regulated function that is initiated by β-adrenergic stimulation and highly coupled to mitochondrial ATP synthesis (24). β-Receptor stimulation results in activation of protein kinase-A, hormone-sensitive lipase (HSL), and adipose triglyceride lipase (ATGL) which leads to conversion of triglycerides to free fatty acids. The morphological changes we find highly suggest that lipolysis is occurring in these tissues; however, we found no difference in fatty acid levels between septic obese and nonobese mice as both groups had decreased levels compared with baseline. Nonobese septic mice had lower plasma triglyceride levels than obese septic mice. It is possible that septic nonobese mice have lower triglyceride levels because of increased utilization in other tissues. Alternatively, obese mice may have impaired utilization during sepsis. The fact that obese mice have a larger volume of WAT can potentially lead to an unintentional underestimation of the extent of lipolysis and browning in the obese groups. To minimize this, we sampled several areas from each specimen and used different methods to confirm our findings. Human data demonstrate that people with obesity have evidence of impaired catecholamine-induced lipolysis (25), but other studies show that there is no relationship between obesity status and fasting fatty acid levels (26). Taken together, given the energy demands during sepsis, adipocyte shrinkage and lipolysis would prove beneficial to use and breakdown lipids for fuel. But this process is impaired in obese septic mice. Future studies will be necessary to determine whether the morphologic appearance of lipolysis affects metabolic function of the adipose tissue.

In addition to adipocyte breakdown, the number of mitochondria increases in nonobese mice during sepsis but not in obese mice. The increased mitochondrial number may be a mechanism to increase available energy in WAT. Lipid droplet breakdown and lipolysis may provide available energy to the organism and may explain the close physical proximity seen between lipid droplets and mitochondria. However, the interaction between these two structures needs further exploration.

Recent evidence suggests browning occurs in WAT in patients with burn injury and cancer-associated cachexia (16, 27), but until now has not been described in sepsis. To the best of our knowledge, this is the first study to demonstrate that browning occurs in sepsis. Crowell et al. demonstrated WAT UCP1 expression increased during recovery from sepsis (28). We expand on Crowell's findings and confirm an increase in UCP1 expression using three different modalities (IHC, Western blot, and ELISA). The increase in UCP1 expression coincided with changes in adipocyte morphology, findings suggestive of browning.

The mechanisms that lead to browning in sepsis remain unknown. One possible initiator of browning is inflammation. It is unclear whether UCP1 expression and browning are dependent on inflammatory cell infiltration. In the present study, adipose tissue neutrophil infiltration, as evidenced by increased MPO, occurred in eWAT from nonobese septic but not obese septic mice. Previous data from our laboratory also demonstrate that during sepsis, obese mice have a diminished adipose tissue inflammatory response including lower plasma IL-6 levels in early sepsis (29, 30). IL-6 is important for browning (31). Using a chimeric mouse burn model, Abdullahi et al. demonstrate bone marrow-derived IL-6 is the main regulator of browning (31). Taken together, these findings suggest that the lack of inflammatory response in obese septic mice may explain the failure of browning.

Unlike brown adipocytes who express high levels of UCP1 under basal (unstimulated) conditions, WAT-induced browning occurs in response to norepinephrine stimulation (32). BAT is highly vascularized and its activation is regulated by the sympathetic nervous system via β-adrenergic receptors (33). Adrenergic stress increases UCP1 expression in both BAT and WAT (11). We hypothesize that since sepsis causes a catecholamine surge and a massive sympathetic response with stimulation of β-adrenergic receptors, it is this process that may initiate WAT browning during sepsis. A limitation to our study is that we did not provide direct measurements of catecholamines and catecholamine receptors. Hahn et al. used a polymicrobial animal model of sepsis, and found elevated levels of catecholamines as early as 5 h after CLP and levels remained elevated during the acute phase of sepsis (34). Obesity is accompanied by a decrease in β-adrenergic receptor subtypes (35) which could explain, in part, the lack of browning found in obese adipose tissue after sepsis. Future experiments blocking catecholamine receptors may be beneficial and provide additional information clarifying the exact mechanism by which browning occurs in sepsis but are blunted in obesity.

One of the fundamental questions is what advantage, if any, does the browning process provide to the organism? It is possible that browning is merely a way to regulate temperature through increased expression of UCP1 and heat production. Browning may also provide an advantage in the way energy is produced and used, given the increased number of mitochondria near multilocular lipid droplets. It is possible that the impaired ability of obese mice to undergo browning contributes to higher mortality. More studies are warranted to understand these processes.

In conclusion, we demonstrate that sepsis induces changes in WAT that are consistent with browning. We consider these changes to be an appropriate physiologic response in nonobese mice to sepsis to provide available energy. However, these changes do not occur in obese mice. Impairment of browning could be an explanation for the higher mortality found in obese mice during sepsis. Further studies will be necessary to pursue these investigations.

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Keywords:

Browning; lipolysis; obesity; sepsis; white adipose tissue

© 2018 by the Shock Society