Salsalate Activates Brown Adipose Tissue in Mice
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1544 Diabetes Volume 64, May 2015 Andrea D. van Dam,1,2 Kimberly J. Nahon,1,2 Sander Kooijman,1,2 Susan M. van den Berg,3 Anish A. Kanhai,1,2 Takuya Kikuchi,1,2 Mattijs M. Heemskerk,2,4 Vanessa van Harmelen,2,4 Marc Lombès,5 Anita M. van den Hoek,6 Menno P.J. de Winther,3 Esther Lutgens,3,7 Bruno Guigas,8,9 Patrick C.N. Rensen,1,2 and Mariëtte R. Boon1,2 Salsalate Activates Brown Adipose Tissue in Mice Diabetes 2015;64:1544–1554 | DOI: 10.2337/db14-1125 Salsalate improves glucose intolerance and dyslipid- Salsalate is a nonsteroidal anti-inflammatory drug belong- emia in type 2 diabetes patients, but the mechanism is ing to the salicylate class of drugs. Salicylates are originally still unknown. The aim of the current study was to derived from plants, in which they function as part of the unravel the molecular mechanisms involved in these immune system to combat infections. Nowadays, synthetic beneficial metabolic effects of salsalate by treating mice compounds that break down to salicylates in vivo, including with salsalate during and after development of high-fat aspirin and salsalate, have largely replaced salicylate (1). In – diet induced obesity. We found that salsalate attenu- humans, salicylates have strong anti-inflammatory effects, – ated and reversed high-fat diet induced weight gain, and have therefore been applied in the clinic for several in particular fat mass accumulation, improved glucose decades to treat pain and inflammation caused by rheuma- tolerance, and lowered plasma triglyceride levels. toid arthritis (2,3). Mechanistically, salsalate selectively promoted the up- Studies have shown that salicylates exhibit beneficial take of fatty acids from glycerol tri[3H]oleate-labeled metabolic effects as well. A recent trial (4) has demon- lipoprotein-like emulsion particles by brown adipose tis- METABOLISM strated that salsalate lowers the levels of HbA ,fasting sue (BAT), decreased the intracellular lipid content in 1c BAT, and increased rectal temperature, all pointing to blood glucose, and circulating triglycerides (TGs) in type more active BAT. The treatment of differentiated T37i 2 diabetes patients. Furthermore, salsalate increases energy brown adipocytes with salsalate increased uncoupled expenditure (EE) in human subjects (5). In contrast to respiration. Moreover, salsalate upregulated Ucp1 ex- aspirin, salsalate is not associated with an increased risk pression and enhanced glycerol release, a dual effect of gastrointestinal bleeding and therefore is relatively safe that was abolished by the inhibition of cAMP-dependent for long-term clinical experience. Thus, it is considered protein kinase (PKA). In conclusion, salsalate activates a promising antidiabetic drug (3). The mechanisms un- BAT, presumably by directly activating brown adipo- derlying the beneficial metabolic effects of salsalate re- cytes via the PKA pathway, suggesting a novel mecha- main largely unknown, partly because its receptor has nism that may explain its beneficial metabolic effects in not yet been identified (6). Salicylates have been shown type 2 diabetes patients. to activate AMPK in liver, muscle, and white adipose 1Department of Endocrinology and Metabolic Diseases, Leiden University Medical 9Department of Parasitology, Leiden University Medical Center, Leiden, the Center, Leiden, the Netherlands Netherlands 2 Einthoven Laboratory for Experimental Vascular Medicine, Leiden, the Corresponding author: Andrea D. van Dam, [email protected]. Netherlands Received 23 July 2014 and accepted 24 November 2014. 3Department of Medical Biochemistry, Academic Medical Center, Amsterdam, the Netherlands This article contains Supplementary Data online at http://diabetes 4Department of Human Genetics, Leiden University Medical Center, Leiden, the .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-1125/-/DC1. Netherlands A.D.v.D. and K.J.N. contributed equally to this study. 5Institut National de la Santé et de la Recherche Médicale, Unité 693, Le Kremlin- P.C.N.R. and M.R.B. contributed equally to this study. Bicêtre, France fi 6Department of Metabolic Health Research, Netherlands Organisation for Applied M.R.B. is currently af liated with the Department of Human Biology, Maastricht Scientific Research, Leiden, the Netherlands University, Maastricht, the Netherlands. 7Institute for Cardiovascular Prevention, Ludwig Maximilian’s University Munich, © 2015 by the American Diabetes Association. Readers may use this article as Munich, Germany long as the work is properly cited, the use is educational and not for profit, and 8Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the work is not altered. the Netherlands diabetes.diabetesjournals.org van Dam and Associates 1545 tissue (WAT), suggesting a role for this energy-sensing ki- Glucose was measured using an enzymatic kit from nase in the mechanism of action of the drug (1). However, Instruchemie (Delfzijl, the Netherlands), and insulin was salicylate still improves glucose metabolism in mice lacking measured by ELISA (Crystal Chem Inc., Downers Grove, IL). b the regulatory AMPK- 1 subunit, suggesting involvement Intravenous Glucose Tolerance Test of other pathways as well (1). Mice were fasted for 6 h, a baseline blood sample was The objective of the current study was to investigate obtained, and mice were intravenously injected with 10 the mechanisms underlying the beneficial effects of mL/g body weight glucose dissolved in PBS (200 mg/mL). salsalate on lipid and glucose metabolism by treating Additional blood samples were taken at t = 5, 15, 30, 60, APOE*3-Leiden.CETP (E3L.CETP) transgenic mice, a well- 90, and 120 min. Capillaries were placed on ice and centri- established model for human-like lipoprotein metabolism fuged, and glucose levels were measured as described (7–9), with salsalate mixed through the high-fat diet above. (HFD). We found that salsalate both prevented and re- duced HFD-induced weight gain by lowering fat mass ac- In Vivo Clearance of Radiolabeled Lipoprotein-Like cumulation, and improved glucose and lipid metabolism. Emulsion Particles Mechanistic studies showed that these effects were ac- Lipoprotein-like TG-rich emulsion particles (80 nm) labeled 3 3 companied by increased activity of brown adipose tissue with glycerol tri[ H]oleate (triolein, [ H]TO) were prepared (BAT). Taken together, our data indicate that BAT may and characterized as described previously (13). Mice were contribute to the beneficial effects of salsalate on lipid fasted for 6 h (from 7:00 A.M. to 1:00 P.M.) and injected with m and glucose metabolism, and corroborate previous find- 200 L of emulsion particles (1.0 mg TG per mouse) via the ings that targeting BAT may be a valuable strategy for tail vein (t =0).After15min,micewerekilledbycervical correcting metabolic derangements. dislocation and perfused with ice-cold PBS through the heart. Thereafter, organs were harvested and weighed, and RESEARCH DESIGN AND METHODS the uptake of [3H]TO-derived radioactivity was quantified Mice, Diet, and Salsalate Treatment and expressed per gram of wet tissue weight. – E3L.CETP mice were obtained as previously described (7 Rectal Temperature Measurement 9). To assess the effect of salsalate on progression of obe- Rectal temperature was measured between 3:00 and 4:00 sity, dyslipidemia, and hyperglycemia, 10-week-old male P.M. using a rectal probe attached to a digital thermometer E3L.CETP mice were randomized to receive an HFD (BAT-12 Microprobe Thermometer; Physitemp, Clifton, NJ). (45% kcal lard fat; Research Diets) without or with 0.5% (weight for weight [w/w]) salsalate (2-carboxyphenyl salic- Histology ylate; TCI Europe N.V.) for 12 weeks. To assess the effect of Interscapular BAT (iBAT) and gonadal WAT (gWAT) were fi salsalate on the regression of obesity and associated met- removed, xed in 4% paraformaldehyde, dehydrated in fi abolic disorders, 10-week-old, male, diet-induced (12 weeks 70% EtOH, and embedded in paraf n. Hematoxylin-eosin on HFD) obese E3L.CETP mice received salsalate (0.5% w/w) (H-E) staining was done using standard protocols. The fi for 4 weeks. To investigate the effect of salsalate indepen- area of intracellular lipid vacuoles in BAT was quanti ed dent of the E3L.CETP background in a progression set- using ImageJ (National Institutes of Health). ting, 10-week-old, male, wild-type (WT) mice (C57BL/6J Quantification of TG Content in BAT background; Charles River Laboratories) were randomized Lipids were extracted from BAT following a protocol to receive an HFD without or with salsalate for 4 weeks. modified from Bligh and Dyer (14). BAT samples (~50 mg) Mice were individually housed at 21 or 28°C (WT mice). were homogenized in 10 mL ice-cold CH OH/mg tissue. To mask the bitter taste of salsalate, anise (3.33% w/w) 3 Lipids were extracted into an organic phase by the addi- was added to the diet of both groups in all studies (10). tion of 1,800 mLCHOH:CHCl (1:3 volume for volume) Mouse experiments were performed in accordance with the 3 3 to 45 mL homogenate and subsequent centrifugation. The Institute for Laboratory Animal Research Guide for the lower organic phase was evaporated, lipids were resus- Care and Use of Laboratory Animals and have received pended in 2% Triton X-100, and TG content was assayed approval from the University Ethical Review Board (Leiden (see above). BAT lipids were reported per milligram of University Medical Center, Leiden, the Netherlands). protein (BCA Protein Assay Kit; Pierce). Body Weight and Body Composition Measurements Isolation of Stromal Vascular Fraction and Flow Body weight was measured with a scale, and body Cytometry composition was measured using an EchoMRI-100 ana- The gWAT was removed, rinsed in PBS, and minced. lyzer (EchoMRI, Houston, TX). Tissues were digested in a collagenase mixture (DMEM Determination of Plasma Parameters with 20 mmol/L HEPES, Collagenase XI, and Collagenase Upon randomization and at 4-week intervals during I; Sigma) for 45 min at 37°C and passed through a 70-mm treatment, blood samples were collected after a 6-h fast nylon mesh.