Salsalate (Salicylate) Uncouples Mitochondria, Improves Glucose Homeostasis, and Reduces Liver Lipids Independent of AMPK-β1

Abstract

Salsalate is a prodrug of salicylate that lowers blood glucose in patients with type 2 diabetes (T2D) and reduces nonalcoholic fatty liver disease (NAFLD) in animal models; however, the mechanism mediating these effects is unclear. Salicylate directly activates AMPK via the β1 subunit, but whether salsalate requires AMPK-β1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-β1-knockout (AMPK-β1KO) mice were treated with a salsalate dose resulting in clinically relevant serum salicylate concentrations (~1 mmol/L). Salsalate treatment increased VO 2 , lowered fasting glucose, improved glucose tolerance, and led to an ~55% reduction in liver lipid content. These effects were observed in both WT and AMPK-β1KO mice. To explain these AMPK-independent effects, we found that salicylate increases oligomycin-insensitive respiration (state 4o) and directly increases mitochondrial proton Corresponding author: Gregory R. Steinberg, gsteinberg@mcmaster.ca. Duality of Interest. No potential conflicts of interest relevant to this article were reported.Author Contributions. B.K.S. and G.R.S. designed research studies, analyzed data, and wrote the manuscript. B.K.S., R.J.F., E.M.D., A.E.G., M.C.H., V.P.H., E.A.D., K.M., J.D.C., E.P.M., and C.G.R.P. conducted experiments and analyzed data. B.K.S., R.J.F., E.M.D., A.E.G., V.P.H., E.A.D., K.M., J.D.C., E.P.M., B.E.K., M.A.T., and G.R.S. edited the manuscript. G.R.S. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0564/-/DC1. Diabetes. Author manuscript; available in PMC 2017 January 12.Published in final edited form as:Diabetes. 2016 November ; 65(11): 3352-3361. doi:10.2337/db16-0564. CIHR Author Manuscript CIHR Author Manuscript CIHR Author Manuscriptconductance at clinical concentrations. This uncoupling effect is tightly correlated with the suppression of de novo lipogenesis. Salicylate is also able to stimulate brown adipose tissue respiration independent of uncoupling protein 1. These data indicate that the primary mechanism by which salsalate improves glucose homeostasis and NAFLD is via salicylate-driven mitochondrial uncoupling.Nonalcoholic fatty liver disease (NAFLD) is considered an important contributing factor to the development of insulin resistance and type 2 diabetes (T2D) (1). Despite the rising prevalence of NAFLD and importance for the development of T2D, there are currently no pharmacological approaches for the treatment of this disease (2).Salsalate is a prodrug of salicylate and is hydrolyzed in the small intestine to produce two molecules of salicylate (3,4). The circulating concentration of salicylate in humans administered salsalate in T2D clinical trials is ~1 mmol/L (5-8). Salsalate has also been shown to improve symptoms of NAFLD (9) and nonalcoholic steatohepatitis in...

The Role of the Immune System in Metabolic Health and Disease

“…Blockade of IL-1b signaling improved insulin secretion and glucose homeostasis and modestly reduced hemoglobin A1c and fasting glucose levels (Cavelti-Weder et al, 2012). Salicylates, which were shown over a century ago to reduce glucose levels of diabetic patients by inhibiting the IKKb/NF-kB axis (Kopp and Ghosh, 1994;Pierce et al, 1996;Smith et al, 2016;Williamson, 1901;Yuan et al, 2001), lowered HbA1c levels and improved glycemic markers in T2DM patients (Goldfine et al, 2010). However, in follow-up human studies, salicylate treatment had a positive effect only in a subset of diabetic patients (Hundal et al, 2002).…”

Salicylate enhances the response of prostate cancer to radiotherapy

“…Effects of the salicylate dimer, salsalate, and RT in vivo were examined in PC3 xenografts. Grafted animals were provided chow diet without or with 2.5 g/kg salsalate (achieving circulating salicylate levels of 900 μM) and/or treated with RT (10 Gy) (Figure A). In non‐irradiated tumors, salsalate did not alter tumor growth kinetics (Figure B).…”

“…Since clinical trials showed that serum SAL levels of approximately 1 mM can be achieved in human circulation with well‐tolerated oral doses of salsalate, we fed immunocompromised animals grafted with PC3 tumors with salsalate‐supplemented chow diet (2.5 g/kg), known to achieve in mice serum SAL levels of 900 μM …”

“…Since clinical trials showed that serum SAL levels of approximately 1 mM can be achieved in human circulation with well-tolerated oral doses of salsalate, [40][41][42] we fed immunocompromised animals grafted with PC3 tumors with salsalate-supplemented chow diet (2.5 g/kg), known to achieve in mice serum SAL levels of 900 μM. 34 Despite the observed inhibition of proliferation and clonogenic survival of PrCa cells in-vitro by SAL treatment alone, we could not observe suppression of PC3 xenograft growth in animals treated with salsalate diet alone. Currently, we do not have an explanation for this observation.…”

New and revisited approaches to preserving the reperfused myocardium

“…Highly polarized Δψ m is observed in mitochondria that are ‘idling’ (that is, mitochondrial respiration with little to no ATP turnover), suggesting that slight uncoupling might protect mitochondria by reducing ROS formation 95 . Although this strategy has been used to treat other diseases influenced by mitochondrial dysfunction 96 , mild uncoupling in the heart did not markedly reduce mitochondrial ROS production 97 , and actually led to increased ROS production in isolated myocytes 98 . Numerous studies have shown that cardioprotection is associated with sustained, polarized Δψ m ; this energized Δψ m is essential during metabolic insult to replenish endogenous antioxidants via NADPH-linked reactions, which allows for the downstream detoxification of free radicals 99,100 .…”