2784 Diabetes Volume 65, September 2016 Simon A. Hawley,1 Rebecca J. Ford,2 Brennan K. Smith,2 Graeme J. Gowans,1 Sarah J. Mancini,3 Ryan D. Pitt,2 Emily A. Day,2 Ian P. Salt,3 Gregory R. Steinberg,2 and D. Grahame Hardie1 The Na+/Glucose Cotransporter Inhibitor Canagliflozin Activates AMPK by Inhibiting Mitochondrial Function and Increasing Cellular AMP Levels Diabetes 2016;65:2784–2794 | DOI: 10.2337/db16-0058 Canagliflozin, dapagliflozin, and empagliflozin, all recently transporters that carry glucose across apical membranes approved for treatment of type 2 diabetes, were derived of polarized epithelial cells against concentration gradi- from the natural product phlorizin. They reduce hypergly- ents, driven by Na+ gradients. SGLT1 is expressed in the cemia by inhibiting glucose reuptake by sodium/glucose small intestine and responsible for most glucose uptake cotransporter (SGLT) 2 in the kidney, without affecting across the brush border membrane of enterocytes, whereas intestinal glucose uptake by SGLT1. We now report that SGLT2 is expressed in the kidney and responsible for most fl canagli ozin also activates AMPK, an effect also seen glucose readsorption in the convoluted proximal tubules. with phloretin (the aglycone breakdown product of The first identified SGLT inhibitor was a natural product, fi phlorizin), but not to any signi cant extent with dapagli- phlorizin, which is broken down in the small intestine to flozin, empagliflozin, or phlorizin. AMPK activation oc- phloretin, the aglycone form (Fig. 1). Although phlorizin curred at canagliflozin concentrations measured in had beneficial effects in hyperglycemic animals (2), it in- human plasma in clinical trials and was caused by hibits SGLT1 and SGLT2, causing adverse gastrointestinal inhibition of Complex I of the respiratory chain, leading effects (3). This led to development of the synthetic ana- to increases in cellular AMP or ADP. Although canagli- fl fl fl flozin also inhibited cellular glucose uptake indepen- logs canagli ozin (4), dapagli ozin (5), and empagli ozin dently of SGLT2, this did not account for AMPK (6) (Fig. 1), which have 260-, 1,100-, and 2,700-fold se- activation. Canagliflozin also inhibited lipid synthesis, an lectivity for SGLT2 over SGLT1, respectively (6). In meta- fl PHARMACOLOGY AND THERAPEUTICS effect that was absent in AMPK knockout cells and that analyses of clinical trials in type 2 diabetes, canagli ozin required phosphorylation of acetyl-CoA carboxylase (7), dapagliflozin (8), or empagliflozin (9), as monother- (ACC) 1 and/or ACC2 at the AMPK sites. Oral adminis- apy or combined with existing therapies, all reduced fast- tration of canagliflozin activated AMPK in mouse liver, ing plasma glucose, HbA1c, and body weight. Canagliflozin although not in muscle, adipose tissue, or spleen. also decreased plasma triglycerides (7). Because phosphorylation of ACC by AMPK is known to The current front-line therapy for type 2 diabetes is lower liver lipid content, these data suggest a potential metformin, a biguanide that lowers plasma glucose pri- additional benefit of canagliflozin therapy compared with marily by reducing hepatic glucose production (10). Met- other SGLT2 inhibitors. formin, and the related biguanide phenformin, inhibit Complex I of the respiratory chain (11,12) and activate A recently introduced approach to treatment of type 2 the cellular energy sensor AMPK (13,14). Binding to the diabetes is selective inhibition of sodium/glucose co- AMPK-g subunit of AMP and/or ADP, which are elevated transporter (SGLT) 2 (1). SGLT1 and SGLT2 are related during cellular energy stress, causes conformational changes 1Division of Cell Signalling and Immunology, School of Life Sciences, University of This article contains Supplementary Data online at http://diabetes Dundee, Dundee, Scotland, U.K. .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0058/-/DC1. 2 Division of Endocrinology and Metabolism, Department of Medicine, McMaster S.A.H. and R.J.F. contributed equally to this study. University, Hamilton, Ontario, Canada © 2016 by the American Diabetes Association. Readers may use this article as 3Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary long as the work is properly cited, the use is educational and not for profit, and and Life Sciences, University of Glasgow, Glasgow, Scotland, U.K. the work is not altered. More information is available at http://diabetesjournals Corresponding authors: D. Grahame Hardie, [email protected], and .org/site/license. Gregory R. Steinberg, [email protected]. Received 12 January 2016 and accepted 25 May 2016. diabetes.diabetesjournals.org Hawley and Associates 2785 (#325510) was from Abcam, and anti-SGLT2 (sc-47402) was from Santa Cruz Biotechnology. Cell Culture and Lysis HEK-293 cells and wild-type (WT) and AMPK knockout mouse embryo fibroblasts (MEFs) (25) were grown in DMEM with 25 mmol/L glucose and 10% FBS. Cell lysates were prepared as described previously (19). For Western blots shown in Fig. 6C, tissues were homogenized in 5 vols of HES buffer (20 mmol/L Na HEPES [pH 7.4], 1 mmol/L EDTA, 250 mmol/L sucrose; Roche complete protease in- Figure 1—Structures of compounds used in this study. hibitor cocktail) with a Dounce homogenizer and centri- fuged (7,050g, 20 min, 4°C). The pellet was resuspended in HES buffer and layered on top of buffer containing 1.12 mol/L sucrose before centrifugation in a swing-out that activate the kinase via allosteric effects and promotion rotor (41,500g, 60 min, 4°C). Membranes were collected of net phosphorylation of Thr172 on the AMPK-a subunit from the interface of the sucrose layers, diluted in HES (15–18). Metformin and phenformin increase ADP-to-ATP buffer, and centrifuged (150,000g, 60 min, 4°C). The re- ratios and fail to activate AMPK containing a g-subunit sultant plasma membrane–rich pellets were resuspended mutant that does not bind AMP/ADP (19), confirming in HES buffer (0.2–0.4 mL). that their AMPK-activating effects are mediated by in- creases in AMP/ADP. Once activated, AMPK acts to restore Immunoprecipitate Kinase Assays and Other Analyses energy homeostasis by promoting catabolic pathways, in- Methods for AMPK assay in immunoprecipitates, SDS- cluding fatty acid oxidation, while inhibiting anabolic path- PAGE, Western blotting, and determination of cellular ways, including fatty acid synthesis (15,16). Its opposing ADP-to-ATP ratios and oxygen consumption in HEK-293 acute effects on fat synthesis and oxidation are due to cells were described previously (19). Lipid synthesis in phosphorylation of two acetyl-CoA carboxylase (ACC) iso- MEFs was analyzed by starving cells of serum for 3 h forms, ACC1 and ACC2. Whether AMPK explains all ther- and then treating with drug or vehicle in the presence apeutic benefits of metformin has been controversial of [14C]acetate (1 mCi/mL)/0.4 mmol/L Na acetate for because its acute effects on hepatic glucose production in 3 h. Cells were washed with PBS before extraction to de- mice were reported to be AMPK independent (20,21). How- termine incorporation of label into total lipid (26). Fatty 3 ever, studies using knock-in mice, in which both ACC iso- acid oxidation was measured as etomoxir-sensitive H2O forms were replaced by mutants lacking the critical AMPK production from [3H]palmitate. MEFs were preincubated phosphorylation sites, suggested that the longer-term with AMPK activators for 30 min before incubation in insulin-sensitizing effects of metformin are accounted for [3H]palmitic acid (8 mCi/mL, 110 mmol/L), carnitine by phosphorylation and inactivation of ACC1/ACC2 by (50 mmol/L), fatty acid–free BSA (0.5 mg/mL) in Earle’s- AMPK (22). HEPES (116 mmol/L NaCl, 5.3 mmol/L KCl, 0.8 mmol/L fl We now report that canagli ozin activates AMPK, in MgSO4, 1.8 mmol/L CaCl2, 1 mmol/L NaH2PO4,20mmol/L intact cells and in vivo, by a mechanism involving in- HEPES-NaOH, pH 7.4) in the presence or absence of eto- 3 hibition of respiratory chain Complex I. Our results raise moxir (50 mmol/L) for 90 min at 37°C. The H2O gener- the possibility that some therapeutic benefits of canagli- ated was separated and quantified as previously described flozin might occur via AMPK activation rather than SGLT2 (27). inhibition. Animal Experiments RESEARCH DESIGN AND METHODS All animal procedures were approved by the McMaster Materials and Antibodies University Animal Ethics Research Board. Male and female Canagliflozin, dapagliflozin, and empagliflozin were from mice (16–20 weeks), WT or ACC1/ACC2 (S79A/S212A) Selleck Chemicals, and phlorizin, phloretin, metformin, double knock-in (DKI), were housed in a pathogen-free fa- phenformin, AICAR, and 2,4-dinitrophenol (DNP) were cility under a 12-h light/dark cycle at 23°C, with ad libitum from Sigma-Aldrich. A769662 was synthesized as described access to standard chow and water. Primary hepatocytes (23). Antibodies against phosphorylated (p)Thr172 on were generated from WT and DKI mice and the following AMPK-a (pT172, #2531) were from Cell Signaling Tech- day were treated for 4 h with canagliflozin or vehicle before nology.InFig.7,antibodiesagainstpACC(#3661)and assessing AMPK and ACC phosphorylation and lipid syn- total ACC (#3676) were from Cell Signaling Technology. thesis, as previously described (22,28). For experiments to In other figures, total ACC was detected using streptavidin examine AMPK and ACC phosphorylation in vivo, canagli- directly conjugated to 800 nm fluorophore (Rockland Im- flozin or vehicle (saline solution containing 0.5% carboxy- munochemicals), and pACC (14) and total AMPK-a (24) methyl cellulose, 0.025% Tween-20) was administered by antibodies were as previously described. Anti-GLUT1 oral gavage (100 mg/kg, 10 mL/g). Mice were anesthetized 2786 Canagliflozin Activates AMPK Diabetes Volume 65, September 2016 and tissues snap frozen in situ as previously described (22).