Salsalate improves glucose intolerance and dyslipidemia in type 2 diabetes patients, but the mechanism is still unknown. The aim of the current study was to unravel the molecular mechanisms involved in these beneficial metabolic effects of salsalate by treating mice with salsalate during and after development of high-fat diet–induced obesity. We found that salsalate attenuated and reversed high-fat diet–induced weight gain, in particular fat mass accumulation, improved glucose tolerance, and lowered plasma triglyceride levels. Mechanistically, salsalate selectively promoted the uptake of fatty acids from glycerol tri[3H]oleate-labeled lipoprotein-like emulsion particles by brown adipose tissue (BAT), decreased the intracellular lipid content in BAT, and increased rectal temperature, all pointing to more active BAT. The treatment of differentiated T37i brown adipocytes with salsalate increased uncoupled respiration. Moreover, salsalate upregulated Ucp1 expression and enhanced glycerol release, a dual effect that was abolished by the inhibition of cAMP-dependent protein kinase (PKA). In conclusion, salsalate activates BAT, presumably by directly activating brown adipocytes via the PKA pathway, suggesting a novel mechanism that may explain its beneficial metabolic effects in type 2 diabetes patients.
Salsalate is a nonsteroidal anti-inflammatory drug belonging to the salicylate class of drugs. Salicylates are originally derived from plants, in which they function as part of the immune system to combat infections. Nowadays, synthetic compounds that break down to salicylates in vivo, including aspirin and salsalate, have largely replaced salicylate (1). In humans, salicylates have strong anti-inflammatory effects, and have therefore been applied in the clinic for several decades to treat pain and inflammation caused by rheumatoid arthritis (2,3).
Studies have shown that salicylates exhibit beneficial metabolic effects as well. A recent trial (4) has demonstrated that salsalate lowers the levels of HbA1c, fasting blood glucose, and circulating triglycerides (TGs) in type 2 diabetes patients. Furthermore, salsalate increases energy expenditure (EE) in human subjects (5). In contrast to aspirin, salsalate is not associated with an increased risk of gastrointestinal bleeding and therefore is relatively safe for long-term clinical experience. Thus, it is considered a promising antidiabetic drug (3). The mechanisms underlying the beneficial metabolic effects of salsalate remain largely unknown, partly because its receptor has not yet been identified (6). Salicylates have been shown to activate AMPK in liver, muscle, and white adipose tissue (WAT), suggesting a role for this energy-sensing kinase in the mechanism of action of the drug (1). However, salicylate still improves glucose metabolism in mice lacking the regulatory AMPK-β1 subunit, suggesting involvement of other pathways as well (1).
The objective of the current study was to investigate the mechanisms underlying the beneficial effects of salsalate on lipid and glucose metabolism by treating APOE*3-Leiden.CETP (E3L.CETP) transgenic mice, a well-established model for human-like lipoprotein metabolism (7–9), with salsalate mixed through the high-fat diet (HFD). We found that salsalate both prevented and reduced HFD-induced weight gain by lowering fat mass accumulation, and improved glucose and lipid metabolism. Mechanistic studies showed that these effects were accompanied by increased activity of brown adipose tissue (BAT). Taken together, our data indicate that BAT may contribute to the beneficial effects of salsalate on lipid and glucose metabolism, and corroborate previous findings that targeting BAT may be a valuable strategy for correcting metabolic derangements.
Research Design and Methods
Mice, Diet, and Salsalate Treatment
E3L.CETP mice were obtained as previously described (7–9). To assess the effect of salsalate on progression of obesity, dyslipidemia, and hyperglycemia, 10-week-old male E3L.CETP mice were randomized to receive an HFD (45% kcal lard fat; Research Diets) without or with 0.5% (weight for weight [w/w]) salsalate (2-carboxyphenyl salicylate; TCI Europe N.V.) for 12 weeks. To assess the effect of salsalate on the regression of obesity and associated metabolic disorders, 10-week-old, male, diet-induced (12 weeks on HFD) obese E3L.CETP mice received salsalate (0.5% w/w) for 4 weeks. To investigate the effect of salsalate independent of the E3L.CETP background in a progression setting, 10-week-old, male, wild-type (WT) mice (C57BL/6J background; Charles River Laboratories) were randomized to receive an HFD without or with salsalate for 4 weeks.
Mice were individually housed at 21 or 28°C (WT mice). To mask the bitter taste of salsalate, anise (3.33% w/w) was added to the diet of both groups in all studies (10). Mouse experiments were performed in accordance with the Institute for Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and have received approval from the University Ethical Review Board (Leiden University Medical Center, Leiden, the Netherlands).
Body Weight and Body Composition Measurements
Body weight was measured with a scale, and body composition was measured using an EchoMRI-100 analyzer (EchoMRI, Houston, TX).
Determination of Plasma Parameters
Upon randomization and at 4-week intervals during treatment, blood samples were collected after a 6-h fast and assayed for levels of TGs, total cholesterol (TC), and free fatty acids (FFA), as described previously (11,12). Glucose was measured using an enzymatic kit from Instruchemie (Delfzijl, the Netherlands), and insulin was measured by ELISA (Crystal Chem Inc., Downers Grove, IL).
Intravenous Glucose Tolerance Test
Mice were fasted for 6 h, a baseline blood sample was obtained, and mice were intravenously injected with 10 µL/g body weight glucose dissolved in PBS (200 mg/mL). Additional blood samples were taken at t = 5, 15, 30, 60, 90, and 120 min. Capillaries were placed on ice and centrifuged, and glucose levels were measured as described above.
In Vivo Clearance of Radiolabeled Lipoprotein-Like Emulsion Particles
Lipoprotein-like TG-rich emulsion particles (80 nm) labeled with glycerol tri[3H]oleate (triolein, [3H]TO) were prepared and characterized as described previously (13). Mice were fasted for 6 h (from 7:00 a.m. to 1:00 p.m.) and injected with 200 μL of emulsion particles (1.0 mg TG per mouse) via the tail vein (t = 0). After 15 min, mice were killed by cervical dislocation and perfused with ice-cold PBS through the heart. Thereafter, organs were harvested and weighed, and the uptake of [3H]TO-derived radioactivity was quantified and expressed per gram of wet tissue weight.
Rectal Temperature Measurement
Rectal temperature was measured between 3:00 and 4:00 p.m. using a rectal probe attached to a digital thermometer (BAT-12 Microprobe Thermometer; Physitemp, Clifton, NJ).
Interscapular BAT (iBAT) and gonadal WAT (gWAT) were removed, fixed in 4% paraformaldehyde, dehydrated in 70% EtOH, and embedded in paraffin. Hematoxylin-eosin (H-E) staining was done using standard protocols. The area of intracellular lipid vacuoles in BAT was quantified using ImageJ (National Institutes of Health).
Quantification of TG Content in BAT
Lipids were extracted from BAT following a protocol modified from Bligh and Dyer (14). BAT samples (~50 mg) were homogenized in 10 µL ice-cold CH3OH/mg tissue. Lipids were extracted into an organic phase by the addition of 1,800 µL CH3OH:CHCl3 (1:3 volume for volume) to 45 µL homogenate and subsequent centrifugation. The lower organic phase was evaporated, lipids were resuspended in 2% Triton X-100, and TG content was assayed (see above). BAT lipids were reported per milligram of protein (BCA Protein Assay Kit; Pierce).
Isolation of Stromal Vascular Fraction and Flow Cytometry
The gWAT was removed, rinsed in PBS, and minced. Tissues were digested in a collagenase mixture (DMEM with 20 mmol/L HEPES, Collagenase XI, and Collagenase I; Sigma) for 45 min at 37°C and passed through a 70-µm nylon mesh. The suspension was centrifuged (6 min, 1,250 revolutions per minute), and the pelleted stromal vascular fraction (SVF) was resuspended in FACS buffer. Fc blocking (CD16/32 antibody) was performed prior to a 30 min staining with fluorescently labeled primary antibodies for CD45, CD11b, Ly6G, F4/80, CD11c, CD206, Gr-1, CD19, CD3, CD4, CD8, CD25, and FoxP3 (BioLegend, e-Bioscience, or BD Biosciences). SVF was analyzed by flow cytometry with a BD FacsCANTO II flow cytometer and FlowJo software.
Analysis of DNA Content
DNA content in gWAT samples was quantified as previously described (15).
Culture and Differentiation of White and Brown Adipocytes
3T3-L1 (American Type Culture Collection, Manassas, VA) and T37i cells (16,17) were cultured and differentiated as described previously. Differentiated cells were treated with salsalate (SML0070; Sigma), sodium salicylate (S2007; Sigma), AICAR (ab120358; Abcam), norepinephrine (A7257; Sigma), CL316243 (Tocris Bioscience, Bristol, U.K.), or vehicle (DMSO) for 15 min or 8 h. H89 (H-5239, LC Laboratories) was added 1 h before initiation of salsalate treatment. Supernatant was collected for the determination of glycerol concentration (Instruchemie), and cells were harvested for RNA or protein analysis as described below.
Oxygen Consumption Measurements
A Seahorse Bioscience XF96 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA) was used to measure the oxygen consumption rate (OCR) in differentiated T37i cells. On day 8 of differentiation, cells were trypsinized and seeded in a 96-well Seahorse Bioscience assay plate. The next day, the ATP synthase inhibitor oligomycin, vehicle, salsalate, and CL316243 were preloaded in the reagent delivery chambers and pneumatically injected into the wells. Oligomycin was injected to a final concentration of 1.5 μmol/L, and the OCR was measured three times after mixing. Then, vehicle (0.1% DMSO), salsalate, and/or CL316243 were injected, and the OCR was measured six times in 30 min. All OCR measurements were normalized to cell count using Cyquant (Invitrogen).
Western Blot Analysis
Pieces of snap-frozen mouse tissue (~50 mg) or T37i cells (grown in 3.8 cm2 wells) were lysed, protein was isolated, and Western blots were performed as previously described (11). Primary antibodies and dilutions are listed in Supplementary Table 1. Protein content was corrected for a control mix on each blot and for the housekeeping protein tubulin.
RNA Purification and Quantitative RT-PCR
RNA was extracted from snap-frozen mouse tissue (~25 mg) or T37i cells (grown in 3.8 cm2 wells) using Tripure RNA Isolation reagent (Roche). Total RNA (1–2 µg) was reverse transcribed using Moloney Murine Leukemia Virus Reverse Transcriptase (Sigma) for quantitative RT-PCR (qRT-PCR) to produce cDNA. mRNA expression was normalized to β2-microglobulin (β2m) and 36b4 mRNA content and expressed as a fold change compared with control mice using the ΔΔCT method. The primer sequences used are listed in Supplementary Table 2.
All data are expressed as the mean ± SEM, unless stated otherwise. Groups were compared with a two-tailed unpaired Student test and were considered statistically significant if P < 0.05.
Salsalate Prevents HFD-Induced Obesity
To investigate the effect of salsalate on the development of obesity, male E3L.CETP mice were fed an HFD with or without salsalate for 12 weeks (i.e., progression study). Salsalate prevented the HFD-induced increase in body mass seen in the control group (−80%, 1.6 ± 2.6 vs. 7.9 ± 5.5 g, P < 0.01; Fig. 1A), which was due to a decreased gain in fat mass (−60%, 3.1 ± 2.4 vs. 7.8 ± 4.2, P < 0.05; Fig. 1B) rather than lean mass (Fig. 1C). Although salsalate persistently prevented mice from gaining fat mass throughout the treatment period, salsalate decreased food intake only during the first 3 days (Fig. 1D).
Salsalate Prevents HFD-Induced Deterioration of Glucose and TG Metabolism
Next, we assessed whether salsalate protects mice from developing HFD-induced glucose intolerance and dyslipidemia. Although salsalate did not affect fasting glucose levels (Fig. 2A), it reduced insulin levels after 4 weeks (−41%, P < 0.05), 8 weeks (−47%, P = 0.07), and 12 weeks (−67%, P < 0.05; Fig. 2B) of treatment. In addition, salsalate improved glucose tolerance (Fig. 2C), as evidenced by a reduction of the area under the curve (AUC) of glucose concentrations during an intravenous glucose tolerance test (AUC −25%, P < 0.05; Fig. 2D). Salsalate did not affect plasma TC levels (Fig. 2E) throughout the treatment period but decreased plasma TG levels at 8 weeks (−43%, P < 0.01) and 12 weeks (−47%, P < 0.05; Fig. 2F).
To elucidate which metabolic organs were involved in the TG-lowering effect of salsalate, the tissue-specific uptake of fatty acids (FAs) derived from intravenously injected [3H]TO-labeled lipoprotein-like emulsion particles was determined. Salsalate tended to increase the uptake of [3H]TO-derived activity by dorsocervical BAT (dcBAT), although significance was not reached, probably due to the substantial interindividual variation (Fig. 2G).
Salsalate Reverses HFD-Induced Obesity and Improves Glucose and TG Metabolism
We investigated the potential of salsalate to reverse diet-induced obesity in E3L.CETP mice (regression study). In this setting, salsalate lowered body weight (Fig. 3A) mainly due to a reduction in fat mass (Fig. 3B). Furthermore, salsalate reduced fasting glucose (−30%, P < 0.01; Fig. 3C) and TG levels (−46%, P < 0.05; Fig. 3D). Salsalate increased the uptake of [3H]TO-derived activity predominantly by iBAT (156%, P < 0.01) and dcBAT (102%, P < 0.05), and to some extent by the liver (38%, P < 0.01) (Fig. 3E). These data suggest that salsalate activates BAT, and this idea is further supported by the observation that rectal temperature was raised (0.5°C, P < 0.05; Fig. 3F) upon salsalate treatment. Moreover, salsalate reduced iBAT weight (−50%, P < 0.01; Fig. 3G), indicating reduced fat accumulation within the tissue. Indeed, lipid droplet content was lower (Fig. 3H–J), pointing to higher intracellular lipolysis (18).
Salsalate Prevents Body Weight Gain and Activates BAT Irrespective of the Transgenic Background
To exclude that the effects of salsalate were specific to the E3L.CETP transgenic model, male WT mice were fed an HFD with or without salsalate for 4 weeks. We confirmed that salsalate treatment had similar effects on body weight, body composition, and food intake (Supplementary Fig. 1A–D). The prevention of adiposity despite equal food intake suggests that either physical activity or EE is enhanced. Indeed, salsalate increased EE during the dark phase (10%, P < 0.05; Supplementary Fig. 1E). This was especially due to a higher glucose oxidation (29%, P < 0.001; Supplementary Fig. 1F), whereas fat oxidation did not differ (Supplementary Fig. 1G). Accordingly, respiratory exchange ratio was higher during the dark phase (Supplementary Fig. 1H). Physical activity was not affected (Supplementary Fig. 1I), suggesting that the energetic loss is mainly explained by increased EE.
Comparable to our findings in E3L.CETP mice, salsalate reduced plasma insulin levels (Supplementary Fig. 2A) and improved glucose tolerance (Supplementary Fig. 2B–D). This was probably achieved via an insulin-independent mechanism, as no differences in glucose levels were found between the treatment groups upon an insulin challenge (Supplementary Fig. 2E). Of note, AMPK phosphorylation in muscle was enhanced upon salsalate treatment (43%, P < 0.05), as was phosphorylation of its downstream target ACC (acetyl-CoA carboxylase) (75%, P < 0.05; Supplementary Fig. 2F).
In WT mice, salsalate also reduced plasma TG levels (Supplementary Fig. 2G). This was not due to lowered intestinal TG absorption, since salsalate-treated mice did not have significantly lower plasma TG levels in response to an oral olive oil gavage (Supplementary Fig. 2H), and FFA content in feces collected in the third week of treatment was unaltered (Supplementary Fig. 2I). Neither VLDL-TG (Supplementary Fig. 2J) nor VLDL[35S]apolipoprotein B production (Supplementary Fig. 2K) were affected upon 4 weeks of salsalate treatment, further supporting a role for BAT in the TG-lowering effect of salsalate. Indeed, comparable to the effects seen in E3L.CETP mice, salsalate reduced iBAT weight (−42%, P < 0.001; Fig. 4A), lowered lipid content (−29%, P < 0.05; Fig. 4B and C), and tended to increase the uptake of [3H]TO-derived activity by the BAT depots (Supplementary Fig. 3C).
Under thermoneutral conditions, salsalate still prevented the development of HFD-induced obesity (Supplementary Fig. 3A and B). However, thermoneutrality ablated the salsalate-induced tendency toward an increased TG uptake by BAT (Supplementary Fig. 3C) and prevented the reduction in plasma TG levels seen at room temperature (Supplementary Fig. 3D). Thus, noradrenergic input (i.e., slight cold sensing at room temperature) seems necessary to bring about salsalate-induced TG uptake by BAT.
To further elucidate the mechanisms by which salsalate activates BAT and regulates intracellular lipolysis, we measured mRNA and (phosphorylated) protein levels in the BAT of mice housed at 21°C upon treatment. Salsalate did not affect either Ucp1 mRNA expression (data not shown) or uncoupling protein-1 (UCP1) protein content (Fig. 4D). Salsalate tended to increase the PKA-mediated lipolysis-stimulating phosphorylation of hormone-sensitive lipase (HSL) on the Ser563 residue (77%, P = 0.06) but not AMPK-mediated phosphorylation of HSL on the Ser565 residue (19) (Fig. 4D), suggesting increased PKA signaling in BAT. Despite the fact that phosphorylation of ACC, the downstream target of AMPK, was increased, we did not observe significant differences in AMPK expression or phosphorylation (Fig. 4D).
Salsalate Reduces WAT Cell Size and Lowers Inflammation in WT Mice Fed an HFD
Since salsalate massively reduced fat mass, we assessed the phenotype of WAT in more detail. In line with the decreased total fat mass (Figs. 1B and 3B, and Supplementary Fig. 1B), salsalate reduced the weight of the gWAT fat pad (−49%, P < 0.001; Fig. 5A). Histological analysis showed that salsalate reduced adipocyte size (−44%, P < 0.01; Fig. 5B and C), while total cell number, as assessed by DNA content, did not differ (data not shown).
Since salsalate is an anti-inflammatory compound, we investigated the immune cell composition of gWAT by flow cytometry. We did not observe a change in the percentage of CD45+ cells within the SVF, nor in relative monocyte, macrophage, granulocyte, T-cell, and B-cell content (data not shown). However, we found fewer proinflammatory (CD11c+) M1 macrophages and more anti-inflammatory (CD206+) M2 macrophages within the F4/80+ fraction (Fig. 5D and E). Accordingly, salsalate decreased F4/80 and Mcp1 mRNA expression in gWAT (Fig. 5F), confirming fewer proinflammatory macrophages and less recruitment of proinflammatory macrophages (20,21) in the gWAT of salsalate-treated mice. Thus, besides preventing fat accumulation in WAT, salsalate prevented HFD-induced skewing of macrophages toward a proinflammatory phenotype.
Next, we studied whether the diminished adipocyte size after salsalate treatment could be the result of increased TG lipolysis. Phosphorylation levels of HSL on the Ser563 residue (phosphorylated by PKA; Fig. 5G) in gWAT did not differ, nor did plasma FFA levels (data not shown). Moreover, in vitro stimulation of 3T3-L1 cells with salsalate did not influence Ser563-HSL phosphorylation (Supplementary Fig. 4A) and even repressed the glycerol concentration in the supernatant (Supplementary Fig. 4B). Of the oxidation genes we measured in gWAT, only Acc2 was upregulated upon salsalate treatment, suggesting slightly enhanced oxidation in gWAT (Supplementary Fig. 5A). Neither the phosphorylation state nor the expression of AMPK was altered (Fig. 5G) in gWAT. We also investigated markers of lipogenesis in gWAT, but only found an upregulation of Acc1 expression (Supplementary Fig. 5B). Collectively, these data indicate that it is unlikely that lipolysis, oxidation, or lipogenesis in WAT is the primary mechanism responsible for the prevention of fat mass accumulation after salsalate treatment.
Salsalate Directly Activates Brown Adipocytes In Vitro
Since our collective data suggested that the improvement in TG metabolism by salsalate could be due to activation of BAT, we investigated whether salsalate directly activates brown adipocytes and which intracellular mechanisms are involved. Strikingly, the treatment of differentiated T37i adipocytes with salsalate increased uncoupled respiration (Fig. 6A). In line with this, increasing concentrations of salsalate resulted in a dose-dependent increase in Ucp1 expression (up to 227%, P < 0.001; Fig. 6B) as well as glycerol release (up to 92%, P < 0.001; Fig. 6C). Accordingly, salsalate decreased lipid content in the cells, as shown by Nile red staining (−18%, P < 0.05; Supplementary Fig. 6A). Decreased de novo lipogenesis found upon treatment with salsalate (Supplementary Fig. 6B) could also contribute to this finding. Besides Ucp1, salsalate upregulated several other genes involved in BAT function (Fig. 6D).
The majority, but not all, of orally administered salsalate is metabolized to salicylate in vivo, resulting in plasma concentrations of 1–3 mmol/L (1). Like salsalate, salicylate (1 mmol/L) enhanced Ucp1 expression in T37i brown adipocytes (53%, P < 0.01, Supplementary Fig. 6C). Salicylate (1 and 3 mmol/L) also enhanced the expression of Pparα (up to 304%, P < 0.01) and its target gene (22) Elovl3 (up to 224%, P < 0.05; Supplementary Fig. 6C), while the higher dosage of salicylate (3 mmol/L) enhanced glycerol release (80%, P < 0.01; Supplementary Fig. 6D).
Since salsalate is known to directly activate AMPK (1), we investigated this in brown adipocytes. However, salsalate did not increase the phosphorylation of AMPK (Fig. 6E) or ACC (data not shown) after stimulation for 15 min, 1 h, or 8 h (data not shown). We therefore searched for another route by which salsalate may activate BAT.
As both AMPK and PKA regulate lipolysis in BAT by phosphorylation of HSL (23,24), and our in vivo data indicated PKA-mediated phosphorylation of HSL on the Ser563 residue, we investigated whether salsalate activates the PKA-HSL route in brown adipocytes. Salsalate enhanced phosphorylation of the 30-kDa PKA substrate in brown adipocytes after 8 h of stimulation (69%, P < 0.05) and increased phosphorylation of HSL on Ser563, the PKA phosphorylation site of this lipolytic enzyme (108%, P < 0.05; Fig. 6F), while phosphorylation of HSL on Ser565, the AMPK phosphorylation site, was unaffected. To investigate the involvement of the PKA pathway for BAT activation by salsalate, we stimulated brown adipocytes with salsalate in the presence of the PKA inhibitor H89 (25 μmol/L). H89 blunted the salsalate-induced Ser563-HSL phosphorylation (Fig. 6G), upregulation of Ucp1 expression (Fig. 6H), and glycerol release (Fig. 6I), indicating that the PKA pathway is required for brown adipocyte activation by salsalate. These data are consistent with our observation that salsalate only increased TG uptake by BAT in vivo when mice were housed at room temperature (i.e., when adrenergic signaling is present).
Since PKA is a downstream target of β-adrenergic signaling, we investigated whether salsalate acts in synergism with β-adrenergic stimulation in T37i brown adipocytes. Although salsalate in combination with the β3-agonist CL316243 enhanced uncoupled respiration compared with CL316243 alone, no synergistic effect was found (Fig. 6J). Furthermore, salsalate and norepinephrine showed an additive effect on Ucp1 expression (Supplementary Fig. 6E). This suggests that increased β-adrenergic signaling is not required for the effects of salsalate, but since the inhibition of PKA abolishes the effects of salsalate, basal β-adrenergic signaling is required. Taken together, both our in vitro and in vivo data point to an interplay between β-adrenergic and salsalate-induced signaling.
Previous studies (3,4) in humans and animals have shown that salsalate has beneficial metabolic effects by improving glucose tolerance and lowering plasma TG levels. The mechanisms responsible for these changes, however, remained unclear. In the current study, we show that long-term treatment of E3L.CETP and WT mice with salsalate recapitulates these beneficial metabolic effects, and in addition prevents and reduces body weight gain, mainly by lowering fat mass accumulation. Moreover, we provide evidence from both in vivo and in vitro studies that salsalate activates BAT, an important player in energy metabolism. To the best of our knowledge, this is the first study reporting that activation of BAT may, at least partly, underlie the beneficial metabolic effects of salsalate.
The finding that salsalate prevented body weight gain and fat mass accumulation accompanied by a reduced white adipocyte size has been reported before in rats (25). Lower fat accumulation may be the consequence of a lower food intake or higher EE. Although we noticed that food intake was transiently reduced upon salsalate treatment in mice, the preventive effect on fat mass accumulation persisted throughout the studies. We found increased EE in our study, which is in line with studies in humans (2,5) showing that salsalate treatment increases EE by 18%, suggesting that the long-term inhibiting effects of salsalate on body weight gain are caused by increased resting EE.
In our study, salsalate improved glucose tolerance via an insulin-independent mechanism, and lowered plasma glucose and TG levels, which is in line with findings in humans (2,3). Recently, it has been shown (26) that salicylate stimulates phosphorylation of AMPK in the short term and simultaneously increases glucose transport, independent of insulin, into muscle in rat skeletal muscles ex vivo. Correspondingly, in our study, AMPK phosphorylation in muscle was enhanced upon salsalate treatment, as was phosphorylation of its downstream target ACC. Taken together, our data suggest that salsalate improves glucose tolerance by enhancing glucose oxidation and glucose uptake via an insulin-independent pathway. BAT, a metabolically active tissue that contributes to energy metabolism by uncoupling the electron transport chain from ATP synthesis through UCP1, also has a key role in systemic glucose homeostasis by enhancing glucose clearance (27,28). Although we do not have evidence for the possibility that salsalate improves glucose tolerance through BAT activation, we did show that the TG-lowering effect of salsalate was a result of the higher uptake of lipoprotein-TG–derived FA by BAT, as intestinal TG absorption and hepatic VLDL production were unaffected in our experimental settings. We provide strong evidence that BAT was activated upon salsalate treatment, since we found reduced intracellular lipid content and higher rectal temperature in vivo; higher uncoupled respiration, and upregulation of the expression of thermogenic markers Ucp1 and Elovl3; and increased glycerol release upon salsalate treatment in vitro.
Regarding the mechanism through which salsalate activates BAT, we found slightly increased phosphorylation of ACC, the downstream target of AMPK, in BAT in vivo, but not of AMPK itself. In T37i brown adipocytes in vitro, the phosphorylation statuses of AMPK and ACC were also unaffected. Although a recent study by Hawley et al. (1) showed that salicylate activates AMPK in the liver, muscle, and adipose tissue, they also observed that the potency of salicylate to improve fasting glucose and insulin levels, glucose tolerance, and insulin resistance were retained in mice lacking the β1 subunit of AMPK, demonstrating that other pathways are more important than the AMPK-mediated beneficial metabolic effects of salicylates.
Our data point more profoundly toward activation of the PKA-HSL pathway in BAT. We found that salsalate increased Ser563-HSL phosphorylation in BAT in vivo and in brown adipocytes in vitro, which points to an increase of PKA-mediated lipolysis. Indeed, salsalate treatment enhanced glycerol release in vitro and reduced the intracellular lipid content in BAT in vivo. The enhanced intracellular release of FA results in an increased availability of the substrate for oxidation that can also induce allosteric activation of UCP1 (29), both of which result in enhanced uncoupling, which we also demonstrated in brown adipocytes in vitro. Importantly, the fact that PKA inhibition blunted the salsalate-induced Ser563-HSL phosphorylation, Ucp1 expression, and glycerol release in vitro supports a necessity for this pathway in brown adipocyte activation.
Besides improving metabolism, salsalate is an effective anti-inflammatory agent in the clinic. With the current knowledge of a profound link among obesity, inflammation, and type 2 diabetes, we also investigated the effects of salsalate on the inflammatory state of WAT. In gWAT, salsalate lowered Mcp1 expression, an attraction factor of proinflammatory M1 macrophages, and prevented HFD-induced skewing of macrophages toward this phenotype. Previous research (30) showed that high concentrations of salicylates inhibit nuclear factor-κB activity in vitro, a key transcription factor that regulates inflammation. In obese humans, salsalate lowers the inflammatory state, with a 34% reduction in circulating levels of C-reactive protein and a decline in nuclear factor-κB activity in WAT (31,32). This reduction in inflammation might contribute to the improved glucose metabolism upon administration of salsalate.
Since it is becoming increasingly clear that BAT activation and subsequent elevation of EE can lower body fat mass in human adults (33), drugs that target BAT are of great interest in the combat against obesity. Although some human studies (4,34) suggested that salsalate does not affect body weight, the effects on fat mass have not been reported. As mice have a relatively larger amounts of BAT compared with humans (35,36), activation of BAT by salsalate in humans might translate into improved fat distribution, and glucose and lipid metabolism without substantially affecting total body weight.
In conclusion, we show that salsalate exerts beneficial metabolic effects by directly activating BAT through modulation of the PKA pathway in BAT (Fig. 7). Further studies are warranted to investigate whether salsalate activates BAT in humans, thereby preventing obesity and associated disorders.
M.R.B. is currently affiliated with the Department of Human Biology, Maastricht University, Maastricht, the Netherlands.
Acknowledgments. The authors thank Annika Tanke, Ellemiek de Wit, Isabel Mol, Hetty Sips, Trea Streefland, and Chris van der Bent (all from Leiden University Medical Center, Leiden, the Netherlands) for their valuable technical assistance.
Funding. This work was supported by a research grant from the Rembrandt Institute of Cardiovascular Science to M.P.J.d.W., E.L., and P.C.N.R., and by a personal grant of the Board of Directors of Leiden University Medical Center to M.R.B. P.C.N.R. is an Established Investigator of the Dutch Heart Foundation (2009T038).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. A.D.v.D. helped to conceptualize the project, perform the experiments, analyze the data, and draft the manuscript; contributed to the discussion; and critically reviewed the manuscript. K.J.N. helped to perform the experiments, analyze the data, and draft the manuscript. S.K., S.M.v.d.B., A.A.K., T.K., M.M.H., and V.v.H. helped to perform the experiments, analyze the data, and/or critically review the manuscript. M.L. provided the T37i cell line and helped to critically review the manuscript. M.P.J.d.W. and E.L. contributed to the discussion and helped to critically review the manuscript. A.M.v.d.H. and B.G. helped to conceptualize the project, contributed to the discussion, and helped to critically review the manuscript. P.C.N.R. and M.R.B. helped to conceptualize the project and perform the experiments, supervised the project, contributed to the discussion, and edited the manuscript. A.D.v.D., K.J.N., P.C.N.R., and M.R.B. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.