INHIBITION OF LIPOLYSIS WITH ACIPIMOX ATTENUATES POSTBURN WHITE ADIPOSE TISSUE BROWNING AND HEPATIC FAT INFILTRATION

Abstract

Extensive burn injuries promote an increase in the lipolysis of white adipose tissue (WAT), a complication that enhances postburn hypermetabolism contributing to hyperlipidemia and hepatic steatosis. The systemic increase of free fatty acids (FFAs) due to burn-induced lipolysis and subsequent organ fatty infiltration may culminate in multiple organ dysfunction and, ultimately, death. Thus, reducing WAT lipolysis to diminish the mobilization of FFAs may render an effective means to improve outcomes postburn. Here, we investigated the metabolic effects of Acipimox, a clinically approved drug that suppresses lipolysis via inhibition of hormone-sensitive lipase (HSL). Using a murine model of thermal injury, we show that specific inhibition of HSL with Acipimox effectively suppresses burn-induced lipolysis in the inguinal WAT leading to lower levels of circulating FFAs at 7 days postburn (P < 0.05). The FFA substrate shortage indirectly repressed the thermogenic activation of adipose tissue after injury, reflected by the decrease in protein expression of key browning markers, UCP-1 (P < 0.001) and PGC-1α (P < 0.01). Importantly, reduction of FFA mobilization by Acipimox significantly decreased liver weight and intracellular fat accumulation (P < 0.05), suggesting that it may also improve organ function postburn. Our data validate the pharmacological inhibition of lipolysis as a potentially powerful therapeutic strategy to counteract the detrimental metabolic effects induced by burn.

INTRODUCTION

Severe burn injury represents one of the most devastating forms of trauma unrivaled in the extent and duration of debilitation. Patients with thermal injuries that exceed 20% of the total body surface area (TBSA) exhibit a profound hypermetabolic stress response characterized by elevated resting energy expenditure (REE), hyperinflammation, and whole body catabolism (1, 2). Despite improvements in burn care, this pathophysiologic response can leave patients suffering for up to 3 years postinjury (3). The consequences of persistent hypermetabolism, such as hyperlipidemia and hepatic steatosis, remain global health threats with increasing prevalence (4). Effective prevention and treatment strategies, including the option of pharmacological interventions, are needed to halt this detrimental progression. To date, however, only few potent and safe therapeutics that improve hypermetabolic complications are available (5).

From a reductionist point of view, hypermetabolism results from an imbalance between the rates of fat synthesis and fat catabolism in white adipose tissue (WAT) (6). This concept finds strong support in studies showing that increased triglyceride (TG) breakdown and decreased TG synthesis in WAT contribute to the development of cancer-associated cachexia, a condition characterized by severe weight loss and muscle catabolism (7). TG breakdown is defined as the enzymatic cleavage of TGs into free fatty acids (FFAs) and glycerol (8). This process, known as lipolysis, requires at least three distinct hydrolases—adipose TG lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase—which sequentially act to release three FAs from the glycerol backbone (9). Unexpectedly, humans and mice lacking these adipose lipases do not, or only moderately, gain weight (10-12). Therefore, it is unclear whether WAT lipolysis is a major “driver” of the postburn catabolic state.

Although there is an evident correlation between the substantial remodeling of WAT, particularly in the subcutaneous “inguinal” fat, and the induction of both hyperlipidemia and fatty liver, the causative basis for this connection and the role lipolysis plays remains a matter of extensive debate (13). A popular hypothesis proposes that increased WAT lipolysis generates excessive amounts of circulating FFAs, which are subsequently absorbed by the liver and other tissues leading to ectopic fat deposition (14). In this context, postburn hepatic steatosis and tissue dysfunction result from FA-induced lipotoxicity, where FAs undergo transformation into TGs and bioactive lipid species such as diacylglycerols, ceramides, or prostaglandins (15). Recent data from both human and rodent models of burn injury lend credence to this idea.

Indeed, numerous studies have reported that WAT lipolysis is hyperactive in burn patients due to the constitutive activation of ATGL and HSL lipases (16-19). In fact, it was recently revealed that, under prolonged adrenergic stress, WAT adopts brown adipose tissue (BAT) characteristics in a process termed “browning.” This switch, characterized by the presence of uncoupling protein 1 (UCP1), enhances energy expenditure by accelerating WAT breakdown (20). The unrestrained lipolytic response is speculated to further perpetuate postburn hypermetabolism leading to multiple organ failure, sepsis, and, ultimately, death (21). The lipotoxicity model suggests that inhibition of burn-induced lipolysis could be an attractive approach to lower plasma lipid concentrations, thereby reducing the availability of FFAs and the lipotoxic impact on ectopic tissues (22). However, the characterization of mouse models lacking ATGL in specific, or all, tissues of the body discouraged rather than encouraged the development of chemical inhibitors for this enzyme (10, 23). Nonetheless, small-molecule inhibitors for HSL that have been tested in animal models of obesity exhibit astonishingly beneficial metabolic phenotypes, such as improved insulin sensitivity (24). Interestingly, nicotinic acid (niacin), a specific HSL inhibitor, has been clinically prescribed in humans to lower plasma lipids by targeting lipolysis via the G protein-coupled receptor GPR109A (25). By decreasing the concentration of intracellular cyclic AMP (cAMP), cAMP-dependent protein kinase (PKA) is unable to phosphorylate HSL. As such, HSL remains inactive, thereby preventing the breakdown of TG and diminishing the release of FFAs and glycerol into circulation (26).

The present study addressed the question whether inhibition of HSL by the newer, well-tolerated niacin derivative, Acipimox, can prevent or cure burn-induced metabolic derangements. Our findings indicate that inhibitor treatment effectively suppresses WAT lipolysis and improves weight loss, mitochondrial coupling, and hyperlipidemia in mice after burn. The antilipolytic effects of Acipimox also indirectly inhibit hepatic triglyceride synthesis, leading to an observed reduction in fat deposition in the liver after 7 consecutive days of treatment. Thus, drugs similar to Acipimox—that predominantly target adipose tissue lipolysis—may represent an attractive means to treat hypermetabolism and its related metabolic disorders.

MATERIALS AND METHODS

Animals and model

Animal experiments were conducted in accordance and approved by the Sunnybrook Research Institute Animal Care Committee (Toronto, Ontario, Canada). Male wild-type C57BL/6J (WT) mice (7–9 weeks old, n = 5–6/group) were purchased from Jackson Laboratories (Bar Harbor, Maine) and housed at ambient temperature and cared in accordance with the Guide for the Care and Use of Laboratory Animals. All mice were anesthetized with 2.5% isoflurane and shaved along the dorsal spine region. Ringers lactate (2–3 mL) was injected in the dorsal region subcutaneously in all mice to protect the spine and buprenorphine (0.05–0.1 mg/kg body weight) was injected for pain management. A full-thickness, third degree scald burn encompassing 30% TBSA was achieved by immersing the dorsal region of mice in 98°C water for 10 s and the ventral region for 2 s to avoid organ damage. Burned mice were subsequently housed individually in sterile cages and food and water were given ad libitum. Sham mice (control) underwent identical experimental procedures, with the exception of the burn injury. Select mice were treated with a daily i.p. injection of Acipimox (50 mg/kg; Sigma-Aldrich, St. Louis, Mo, CAS# 51037-30-0). We based our dosage on previous murine studies, which have shown efficacy with 50 mg/kg IP QD (27, 28). Mice not receiving Acipimox were given vehicle (saline) injections. All injured rodents were health scored by both certified veterinarians and laboratory staff daily to minimize animal pain and distress. Health scoring was based on a scale of 15, with up to 3 points given for eyes and nose, activity, food intake, grooming, and hydration. Mice were sacrificed on day 7 postburn. The liver and adipose tissue depots (inguinal, epididymal, intrascapular brown) were harvested for further analysis and blood collected after cardiac puncture. All tissues were harvested upon sacrifice and stored in −80°C until analysis.

Histology and immunohistochemistry

Adipose tissue was immediately fixed in 10% formalin and then maintained in 70% ethanol before paraffin embedding. Subsequently, tissues were sectioned and stained with hematoxylin and eosin (H&E) or incubated with UCP1 antibody (Sigma), followed by DAB staining. For Oil Red O, liver tissue was coated with OCT (optimal cutting temperature compound), placed on dry ice and stored at −80°C until further analysis. Frozen tissue blocks were sectioned 10 mm thick, mounted on slides, and fixed in formaldehyde (4%) for 1 min. The slides were stained with Oil Red O for 10 min at room temperature, rinsed with water, and then stained using Gill’s Hematoxylin for 1 min. Imaging was performed on a LSM confocal microscope (Zeiss, Germany).

Western blotting

Proteins from rodent liver and adipose tissue were extracted using RIPA buffer (50 mM Tris-cl pH 7.4, 150 mM NaCl, 1% NP 40, 0.25% Na-deoxycholate, 1 mM PMSF) supplemented with protease (Millipore, #20-201) and phosphatase (Thermo Fisher Scientific, #A32957) inhibitors. BCA assays were used to determine protein concentration and 30 μg of proteins were separated using 10% to 12% sodium dodecyl sulfate (SDS) acrylamide gels. Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked using 5% nonfat skim milk for 1 h, and then washed twice in Tris-buffered saline with Tween (TTBS; 12.1 g Tris, 40 g NaCl, 0.1% Tween 20; pH 7.6) for 5 min. Blots were incubated overnight with the following primary antibodies: 1:1,000 Total HSL (Cell Signaling #4107), 1:1,000 p-HSL^Ser660 (Cell Signaling #4126), 1:1,000 p-HSL^Ser563 (Cell Signaling #4139), 1:1,000 ATGL (Cell Signaling #2138), 1:1,000 UCP-1 (Cell Signaling #14670), 1:1,000 PGC-1α (Cell Signaling #2178), 1:1,000 FAS (Cell Signaling #8023), 1:1,000 Total ACC (Cell Signaling #3676), 1:1,000 p-ACC^Ser79 (Cell Signaling #11818), and 1:5,000 GAPDH (Cell Signaling #5174). The membrane was subsequently washed twice in TBST for 5 min, followed by incubation with secondary antibody, which consisted of horseradish peroxidase-conjugated anti-rabbit antibodies (1:3,000) for 1 h. The membrane was washed in TBST twice for a period of 5 min and detection of the desired protein was achieved with a BioRad ChemiDoc Imaging System and SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Densitometry was performed using ImageJ software.

Biochemical measurements

Plasma FFA levels (Free Fatty Acid Fluorometric Assay Kit; Cayman Chemical), plasma glycerol levels (Glycerol Colorimetric Assay Kit, Cayman Chemical), and liver triglyceride levels (Triglyceride Colorimetric Assay Kit, Cayman Chemical) were quantified using assay kits according to the manufacturer’s instructions.

Statistical analysis

All data is presented as mean ± SEM and was analyzed using GraphPad Prism 6.0 (San Diego, Calif). We calculated the mean and standard deviation of a given data set, and then calculated the cutoff for identifying outliers as more than 2 standard deviations from the mean. Only two outliers were detected and removed from the data sets. These values were confirmed on GraphPad Prism 6 using the ROUT method for identifying outliers. Statistical differences between 3 or more groups were evaluated using a one and two-way ANOVA followed by Bonferroni posttests where indicated, with significance accepted at P < 0.05, P < 0.01, P < 0.001.

RESULTS

Acipimox treatment protects mice from burn-induced weight loss independent of alterations in adipose tissue

As previously described, fat stores are mobilized after burn injury to satisfy the high energy demands that result from the hypermetabolic state. Given that this excessive release of FFA contributes to poor patient outcomes, we investigated the metabolic effects of inhibiting WAT lipolysis on postburn hypermetabolism. To achieve this, we administered the clinically approved drug, Acipimox, for 7 days postburn using a nonlethal murine model of thermal injury. Over the course of treatment, vehicle-treated burn mice lost an average of 0.4 g of body weight per day, whereas total body weight decreased by 1.9% postinjury (Fig. 1A). In contrast, Acipimox administration protected against weight loss induced by severe burn, as treated mice gained 0.21 g of body weight per day with total body weight increasing by 0.8%. The absence of burn-induced weight loss in the treated group at 7 days postinjury can partially be explained by the increased food intake relative to the vehicle-treated burn mice (48.5% vs. 30.1%, P < 0.05) (Fig. 1B). Surprisingly, treatment with the lipolysis inhibitor did not affect fat content after injury (Fig. 1C). When comparing adipose tissue mass, the untreated burn mice lost 1.2% of their epididymal white adipose tissue depot (eWAT), whereas the Acipimox treated group similarly lost 1.0% after burn (P < 0.001). Although no change in the mass of inguinal white adipose tissue (iWAT) was observed, Acipimox treatment seemed to reduce liver weight after injury (Fig. 1D). At 7 days postburn, relative liver weight was significantly elevated in the untreated burn group (5.252% vs. 4.264%, P < 0.001). However, treated burn mice displayed no differences in liver weight relative to their control counterpart, suggesting Acipimox may have decreased ectopic fat accumulation postinjury (4.387% vs. 4.264%, P = 0.9168). To uncover the primary mechanism by which Acipimox exerts its hepatic effects, we investigated its antilipolytic activity in the specific adipose tissue depots after burn.

Fig. 1. Effect of Acipimox on body weight and composition.

(A) Postburn changes in body weight over the course of 7 days. (B) Increased dietary intake as a percentage of total food consumed in Acipimox treated mice compared with controls and vehicle-treated burn mice. (C) Adipose tissue weights expressed as a percentage of body weight demonstrating alterations in iWAT eWAT, and iBAT. (D) Increased liver weight in untreated burn mice relative to control and Acipimox treated mice. Values are presented as mean ± standard error. * P < 0.05; ** P < 0.01, n = 6/group.

Acipimox treatment suppresses burn-induced lipolysis in the iWAT

First, we performed histological analyses of the specific WAT depots from control, burn, and burn mice injected with Acipimox for 7 days. Although no striking morphological differences could be observed in eWAT, we noticed the presence of multilocular adipocytes in the iWAT of burned mice, which was not observed in the treated burn mice. As illustrated in Figure 2A, treatment with Acipimox was able to maintain a larger lipid droplet size at 7 days postburn. Our Western blot data corroborated these findings, indicating lower protein expression of key lipolytic enzymes in the iWAT (Fig. 2B). Postburn treatment for 7 days decreased total HSL and ATGL protein levels in the iWAT (0.4574 vs. 1.101, P < 0.01 for T-HSL; 1.202 vs. 1.765, P < 0.05 for ATGL) (Fig. 2C). The antilipolytic action of Acipimox appeared to be mediated by PKA, which consequently lowered the phosphorylation of HSL at serine 660, thereby reducing its enzymatic activity (0.5639 vs. 3.125, P < 0.001) (Fig. 2C). As blocking WAT lipolysis should diminish FFA mobilization, we next assessed the effects of Acipimox on postburn plasma metabolite concentrations.

Fig. 2. Acipimox suppresses burn-induced lipolysis in the iWAT.

(A) H&E staining of iWAT and eWAT in control, vehicle-treated, and Acipimox-treated burn mice. (B) Representative cropped Western blots for p-HSL ser660, p-HSL ser563, T-HSL, ATGL, and GAPDH in the iWAT at 7 days postburn. (C) Protein expression of p-HSL ser660, p-HSL ser563, T-HSL, ATGL normalized to GAPDH levels in iWAT at 7 days postburn. Values are presented as mean ± standard error. * P < 0.05; ** P < 0.01, n = 6/group.

Acipimox-mediated inhibition of lipolysis reshapes FFA flux postburn

To confirm the observed inhibitory effects of Acipimox on burn-induced WAT lipolysis, we compared plasma FFA concentrations in both untreated and treated burn mice. Indeed, burn mice treated with Acipimox displayed lower plasma FFA concentrations relative to the untreated burn group (289.1 μmol/L vs. 904.7 μmol/L, P < 0.001) (Fig. 3A). In fact, postburn treatment with Acipimox not only restored baseline levels of circulating FFA (289.1 μmol/L vs. 416.6 μmol/L, P = 0.2612), but also displayed a declining trend in the relative plasma glycerol concentrations at 7 days postburn (38.32 mg/L vs. 59.69 mg/L, P = 0.052) (Fig. 3B). Overall, these results indicate that Acipimox affects lipid metabolism by specifically targeting key lipolytic enzymes in the iWAT, ultimately decreasing the excessive release of FFAs after burn injury.

Fig. 3. Acipimox-mediated inhibition of lipolysis reshapes FFA flux postburn.

(A) FFA concentration in control, vehicle-treated, and Acipimox-treated burn mice. (B) Plasma glycerol concentration in control, vehicle-treated, and Acipimox-treated burn mice. Values are presented as mean ± standard error. ** P < 0.01, n = 6/group.

Reduction of FFA mobilization mitigates hepatic fat infiltration postburn

As WAT catabolism, release of FFAs, and organ fatty infiltration are common postburn complications, we sought to investigate the cause of postburn liver weight discrepancies between the untreated and treated burn mice. We hypothesized that the lower liver weight of Acipimox-treated mice is attributed to a reduction in hepatic fat infiltration secondary to decreased WAT lipolysis and circulating FFAs. Indeed, differences in hepatic fatty deposition between the two burn groups were demonstrated and further confirmed with Oil Red O staining for lipid droplets. As shown in Figure 4A, Oil Red O staining revealed the presence of lipids in the liver at 7 days postburn, whereas treatment with Acipimox effectively minimized the extent of fatty infiltration. This prompted us to assess whether reducing circulating FFA levels with Acipimox can in fact alter lipid metabolism in the liver. To assess the effects of FFA substrate shortage on hepatic de novo lipogenesis, we analyzed the rate-limiting step of fatty acid biosynthesis, acetyl-CoA carboxylase (ACC) (Fig. 4B). Compared with the nontreated burn group, Acipimox treatment increased the inhibitory phosphorylation of ACC at serine 79 at 7 days postburn (2.746 vs. 1.778, P < 0.05) (Fig. 4C). This was accompanied by a decrease in fatty acid synthase (FASN) protein expression relative to the control mice, suggesting that postburn Acipimox administration limits structural lipid synthesis (0.483 vs. 1.00, P < 0.001) (Fig. 4, B and C). Consistent with these findings, increased triglyceride accumulation was observed in the liver of untreated mice, whereas lowering the concentration of circulating FFAs with Acipimox decreased liver triglyceride content (247.2 mg/dL vs. 118.4 mg/dL, P < 0.05) (Fig. 4D). Taken together, our results demonstrate that Acipimox treatment effectively reduces burn-induced lipolysis, FFA mobilization, and organ specific de novo lipogenesis. Given that these antilipolytic effects protect from ectopic fat accumulation, our results suggest that it perhaps also mitigates hepatic steatosis, which may prove beneficial for organ function after burn.

Fig. 4. Reduced FFA mobilization via Acipimox mitigates hepatic fat infiltration postburn.

(A) Oil Red O staining for adipose deposition in control, vehicle-treated, and Acipimox-treated burn mice. (B) Representative cropped Western blots for p-ACC, T-ACC, FASN, and GAPDH in the liver at 7 days postburn. (C) Protein expression of p-ACC over T-ACC and FASN normalized to GAPDH in the liver at 7 days postburn. (D) Liver triglyceride content in control, vehicle-treated, and Acipimox-treated burn mice. Values are presented as mean ± standard error. * P < 0.05; ** P < 0.01, n = 6/group.

Pharmacological inhibition of lipolysis ameliorates postburn WAT browning

Previous studies have demonstrated that WAT lipolysis fuels cold-induced thermogenesis, and browning may be a compensatory response to facilitate oxidation of excess circulating FFAs (29). As shown above, Acipimox treatment effectively suppressed postburn WAT lipolysis leading to a decreased presence of multilocular adipocytes, a key feature of beige adipose tissue formation (Fig. 4A). Given the clear interconnection between lipolysis and WAT browning, we subsequently decided to investigate the role of WAT lipolysis in the burn-induced thermogenic activation of iWAT. To that end, we assessed the expression of key browning markers at 7 days postburn in Acipimox treated and untreated iWAT after burn. Given that UCP-1 positivity is an indicator of browning, especially when combined with other anatomical features (30), we proceeded to stain the iWAT for this fat-specific browning marker. As illustrated in Figure 5A, UCP-1 immunostaining indicated the presence of multilocular, UCP-1+ adipocytes in untreated mice at 7 days—characteristics indicative of the phenotypic switch from white to beige adipose. Interestingly, at the same time point, adipocytes in Acipimox treated burn mice exhibit features of WAT rather than beige adipose tissue (e.g., diminished UCP-1 positivity, larger unilocular cells).

Fig. 5. Pharmacological inhibition of lipolysis ameliorates postburn WAT browning.

(A) UCP-1 staining in control, vehicle-treated, and Acipimox-treated burn mice. (B) Representative cropped Western blot for UCP-1 (top), PGC1-α (bottom), and GAPDH with a positive control (iBAT) in the iWAT at 7 days postburn. (C) Protein expression of UCP-1 and PGC1-α normalized to GAPDH in the iWAT at 7 days postburn. Values are presented as mean ± standard error. * P < 0.05; ** P < 0.01, n = 6/group.

Based on our immunohistochemical results, Acipimox treatment seems to ameliorate postburn WAT browning at 7 days. To confirm this, we measured protein levels of the browning markers UCP-1 and PGC-1α (Fig. 5B). Our Western blot data supported these findings revealing elevated protein expression of both markers in untreated burn mice relative to the iWAT of Acipimox treated mice at 7 days postburn (36.22 vs. 3.40, P < 0.001 for UCP-1; 5.06 vs. 0.75, P < 0.01 for PGC-1α) (Fig. 5C). Overall, we show that blocking burn-induced WAT lipolysis decreases circulating FFAs, which are potential substrates for browning. Although further investigation is needed, our results suggest that FFAs are required for activation of UCP-1 after burn. Consequently, we demonstrate pharmacological reduction of WAT lipolysis via Acipimox indirectly decreases the browning response to burn injury.

DISCUSSION

High plasma FA concentrations are a well-established risk factor for the development of postburn hypermetabolic complications, such as insulin resistance and liver steatosis (17-19). Although the mechanistic basis of this association is not entirely clear, inhibition of FA mobilization in WAT represents a rational approach to lower plasma FA concentrations and prevents the development of the aforementioned metabolic disorders. Our present study tested this concept in a murine burn model utilizing the clinically approved lipolysis inhibitor, Acipimox. Daily Acipimox administration effectively diminished FFA release from adipose tissue leading to lower circulating levels at 7 days postburn. By reducing intracellular cAMP, Acipimox prevented PKA-mediated activation of HSL at serine 660 and reduced total HSL and ATGL protein levels in iWAT, shifting the balance from β-oxidation toward lipogenesis. As fatty acid oxidation is required for the thermogenic reprogramming of WAT, Acipimox also improved mitochondrial coupling in the adipose tissue of mice after burn (31, 32). This was reflected by a decrease in protein expression of UCP-1 and the mitochondrial biogenesis marker, PGC-1a, in the iWAT of treated burn mice. To our knowledge, this is the first study connecting lipolysis to the key browning regulator, UCP-1, in burns. Although previous work demonstrated that browning enhances lipolysis, we show here that activation of the key lipolytic enzymes HSL and ATGL in turn regulate UCP-1-mediated thermogenesis, suggesting a possible feedback mechanism (33).

The complementary relationship between lipolysis and browning in turn has downstream metabolic effects at the organ level, with implications in inflammation, infection, and metabolism (22). Intriguingly, Acipimox-mediated inhibition of lipolysis and browning was associated with a beneficial metabolic phenotype. Burn mice treated with Acipimox presented with higher body weight and larger adipocyte size compared with the vehicle-treated burn groups at 7 days postinjury. Consistent with previous studies, stimulation of lipolysis after burn decreased both epididymal and inguinal fat mass, reduced adipocyte size, and resulted in total body weight loss (19-21). Although Acipimox administration prevented burn-induced weight loss and preserved adipocyte size, it surprisingly failed to maintain fat mass. Although it seems counterintuitive that inhibition of lipolysis would lead to reduced WAT mass after burn, similar observations have been reported from clinical studies of Acipimox in nondiabetic, insulin-resistant, obese subjects (24, 34). To address the possibility that Acipimox administration itself elevated body weight, separate groups of mice were treated with Acipimox or vehicle but were not subject to burn injury (i.e., control conditions). Results of this experiment showed that Acipimox administration per se had no effect on body weight or composition, but rather increased food intake. Indeed, nicotinic acid and its analogs have been shown to promote the release of growth hormone which, in turn, induces a feedback action on other appetite-regulating hormones, such as ghrelin and leptin (35). In fact, Acipimox itself has been clinically used in the treatment of severe eating disorders, including bulimia nervosa and anorexia nervosa, for the regulation of food intake, glucose disposal, FFA disarray (36). However, the precise mechanism by which reduced lipolysis regulates feeding behavior is currently unknown and awaits clarification.

The most unexpected finding of this study was the robust effect of Acipimox on liver pathology. The compound substantially reduced postburn liver weight and ectopic fat deposition after 7 days of treatment. Compared with the nontreated burn group, Acipimox-treated mice had 67% less liver fat after injury. Similar protective effects of Acipimox on hepatic lipotoxicity and steatosis have been reported in humans (37). Although Acipimox fails to deplete liver fat in healthy subjects, lowering hepatic fatty acid uptake was shown to improve liver function and insulin sensitivity (38). Our findings suggest that WAT-derived FAs drive hepatic fat accumulation in response to burns, and that interference with FA transport from WAT to the liver can halt this process. This beneficial phenotype in Acipimox-treated mice is diametrically different from the hepatic phenotype observed in various genetic mouse models lacking HSL or ATGL in the liver. In these models, all animals developed liver steatosis to a varying extent (10, 39). Instead, pharmacological inhibition of HSL resembles the phenotype of adipose tissue specific HSL and ATGL knockout mice, suggesting that Acipimox-treated mice retain sufficient HSL activity in the liver to evade hepatosteatosis (24, 40). The actual mechanisms by which decreased lipolysis in WAT reduces hepatic fat accumulation may include reduced substrate delivery to the liver, increased insulin sensitivity affecting FA utilization, and/or decreased expression of hepatic genes known to be regulated by FA-sensitive nuclear receptors. Consistent with the latter possibility, we found that Acipimox drastically reduced activity of several prosteatotic proteins, including FASN, which may contribute to resistance to hepatic fat accumulation in treated burn mice. Moreover, these anti-lipolytic effects increased the inhibitory phosphorylation of ACC, suggesting the liver from treated mice were more adept at using fats as fuel in β-oxidation.

It is important to note that, although this particular study focused on lipolysis and browning in the iWAT, the inguinal and epididymal adipose depots have been reported to exhibit different responses to severe burns (21). Although the iWAT has been shown to contain beige precursor adipocytes, the eWAT does not and is resistant to UCP-1 mediated thermogenesis (29). This could potentially account for the browning-related morphological differences observed using our burn rodent model. In accordance with previous murine studies, there were no signs of adipocyte tissue remodeling or induction of UCP1 expression in the eWAT, despite the formation of multilocular beige adipocytes in the iWAT postburn. Although not the focus of this particular work, the differential postburn responses between the iWAT and eWAT warrant a more detailed structure function analysis. In addition, another important limitation of this study was the intergroup variability in expression levels of lipolytic enzymes, particularly phosphor-HSL. The variation in p-HSL-ser 660 protein expression in Acipimox-treated mice may have resulted from increased HSL enzyme protein levels, the transient nature of Acipimox’s action, or the compensatory activity of alternative, currently unknown, TG hydrolases. As such, although we note a significant decrease in p-HSL-ser 660/T-HSL, the data should be interpreted cautiously at this point. Nevertheless, this limitation may be corrected with a longer time course or by varying the drug concentration. According to previously published studies, the lipid lowering effects of nicotinic acid and its derivatives only last for up to 7 h when administered in rats (41). As expected, phosphorylation levels of HSL were profoundly decreased at 1.5 h after treatment. However, most of the inhibitory effects were reversed after 24 h, supporting the concept that the short-term effects of Acipimox may require continuous administration (41).

In conclusion, our study highlights the critical role of lipolysis in postburn hypermetabolism while also shedding light on the role of FFAs in mediating burn-induced fat thermogenesis and hepatic dysfunction. An important caveat of this study is that here, we utilize Acipimox to delineate the temporal relationship between postburn lipolysis and browning. That is, does lipolysis trigger WAT browning or is it a by-product? The data presented herein provide profound evidence that the relationship between the aforementioned postburn phenomena is a vicious feed forward loop: WAT browning-induced lipolysis causes FFA efflux, which, in turn, enhances oxidative metabolism, thus sustaining WAT browning during the hypermetabolic response. Hence, pharmacological inhibition of lipolysis could be an effective means to ameliorate the detrimental metabolic effects induced by severe burns. Although it would be of particular interest to investigate the clinical feasibility of utilizing antilipolytic drugs in burns, future work regarding clinical efficacy and safety profile in these patients is warranted.