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). AMPK by increasing the cellular AMP-to-ATP ratio (19). Measurements of the respiratory exchange ratio (RER) AMPK activation by canagliflozin, A769662, and berber- were performed in metabolic cages using a protocol (29) ine was associated with increased phosphorylation of Thr172 in which mice were fasted overnight and refed with chow on AMPK and of the primary AMPK site on ACC (pACC) for 2 h before being gavaged with canagliflozin or vehicle at (Fig. 2A; quantified results shown in Supplementary Fig. the time of food withdrawal. In separate experiments, mice 1A and B). Figure 2B shows that the effect of 30 mmol/L were treated as above, blood was collected from a nick in the canagliflozin was rapid, reaching a maximum by 20 min. tail, and glucose was measured using a Roche glucometer. Figure 2C and D compares the effects of canagliflozin, fl fl fl Measurements of Oxygen Uptake/Respiration dapagli ozin, and empagli ozin. Although dapagli ozin fl in Primary Mouse Hepatocytes and empagli ozin both activated AMPK, this required . Mitochondrial respiration was measured by high-resolution concentrations 30 mmol/L, and even at 100 mmol/L, fl respirometry (Oxygraph-2 k; Oroboros Instruments, Inns- their effects were small compared with canagli ozin. Ef- fects on phosphorylation of AMPK and ACC (Fig. 2F–H, bruck, Austria) at 37°C and room air-saturated O2 tension in respiration buffer (MIRO5) containing EGTA (0.5 mmol/L), quantified results in Supplementary Fig. 1C–F) were con- fl MgCl2 (3 mmol/L), K-lactobionate (60 mmol/L), KH2PO4 sistent with this. Because single doses of dapagli ozin (10 mmol/L), HEPES (20 mmol/L), sucrose (110 mmol/L), (20 mg) or empagliflozin (50 mg) produce peak plasma and fatty acid–free BSA (1 g/L). Primary hepatocytes from concentrations of only 1–2 mmol/L (33,34), that these WT mice were generated as described above. The follow- inhibitors would produce significant AMPK activation at ing day they were suspended in 2 mL of respiration buffer, normal therapeutic doses seems unlikely. and 800 mL of the suspension was added to the respira- We also examined the effects of the natural product tion chambers. Digitonin (8.1 mmol/L) was added to per- phlorizin and its aglycone form, phloretin. Interestingly, meabilize the cells, and the assay was initiated 5 min later. phloretin activated AMPK and promoted phosphorylation To measure Complex I–supported respiration, glutamate of AMPK and ACC at concentrations slightly higher than (5 mmol/L), malate (2 mmol/L), and ADP (2.5 mmol/L) canagliflozin. However, phlorizin only affected these parame- were added, and respiration was allowed to reach steady ters marginally at much higher concentrations (Fig. 2E, I,and state. To measure Complex II–supported respiration, rote- J;quantification of blots in Supplementary Fig. 1G and H). none (1.25 mmol/L), succinate (10 mmol/L), and ADP Canagliflozin Activates AMPK by Inhibiting Complex I (2.5 mmol/L) were added, and respiration was allowed to of the Respiratory Chain reach steady state. Drugs were then added to respiring cells To test whether canagliflozin activated AMPK by increas- at the indicated concentrations. ing cellular AMP or ADP, we tested its effects in HEK-293 Measurements of Glucose Uptake cells expressing the WT AMPK-g2 subunit (WT cells) or the Cells were incubated in glucose-free Krebs-Ringer phosphate AMP/ADP-insensitive R531G mutant (RG cells) (19). Can- for 2 h, with AICAR present for the last 1 h or canagliflozin/ agliflozin activated AMPK and promoted its phosphoryla- dapagliflozin for the last 15 min. 2-Deoxyglucose (2DG; 50 tion in WT cells but not in RG cells; similar results were mmol/L, 1mCi/mL) was added, and cytochalasin B-sensitive obtained with phloretin (Fig. 3; quantification of blots 2DG uptake was measured over 10 min (30). in Supplementary Fig. 2). These results suggest that canagliflozin and phloretin activate AMPK by increasing Statistical Analysis cellular AMP or ADP. Significance of differences was assessed using the Student Cellular AMP levels are low and difficult to measure in t test, one-way or two-way ANOVA as appropriate, using cultured cells, so we routinely measure ADP-to-ATP ratios the Sidak test after ANOVA. Values of P , 0.05 were as a surrogate for AMP-to-ATP ratios (17). Increasing con- considered statistically significant. centrations of canagliflozin caused increases in the ADP-to- RESULTS ATP ratio that were significant at 10 and 30 mmol/L, with fl Canagliflozin and Phloretin, but not Dapagliflozin, 30 mmol/L canagli ozin producing an effect similar to Empagliflozin, or Phlorizin, Significantly Activate 10 mmol/L phenformin (Fig. 4A). Similar results were AMPK in HEK-293 Cells obtained with phloretin (Fig. 4B). The increase in the cel- To assess the effect of canagliflozin on AMPK, we initially lular ADP-to-ATP ratio due to canagliflozin was accompa- used HEK-293 cells. A daily 300-mg dose of canagliflozin nied by a reduction of cellular oxygen consumption (Fig. produces a peak plasma concentration in humans of ;10 4C). The effect of 30 mmol/L canagliflozin was smaller, mmol/L (31), so we tested concentrations from 1 to although more rapid, than that of 10 mmol/L phenformin. 30 mmol/L. After incubation for 1 h (Fig. 2A), canagliflo- When the uncoupler 2,4-DNP was added 60 min later, zin increased AMPK activity at concentrations from 1 to there was a large increase in oxygen consumption (repre- 30 mmol/L, with 10 mmol/L giving a similar activation to senting the maximal respiration rate when mitochondria that observed with 300 mmol/L A769662, which activates were not limited by the supply of ADP) in cells treated with AMPK by direct binding between the a- and b-subunits DMSO. However, as canagliflozin concentrations increased, (32), or berberine, a mitochondrial inhibitor that activates the effects of 2,4-DNP were eliminated, suggesting that, diabetes.diabetesjournals.org Hawley and Associates 2787

Figure 2—AMPK activation in HEK-293 cells by canagliflozin and other SGLT2 inhibitors. A: HEK-293 cells were treated for 1 h with the indicated concentrations of canagliflozin, A769662, or berberine as positive controls. The upper panel shows kinase assays of immuno- precipitates prepared using anti–pan a antibodies (mean 6 SD, n = 2). The lower panel shows analysis of AMPK and ACC phosphorylation by Western blotting from duplicate dishes of cells. B: Time course of AMPK activation by 30 mmol/L canagliflozin (mean 6 SD, n = 2). C: Comparison of effects of canagliflozin and dapagliflozin at 1-h incubations (mean 6 SEM, n = 4). *P < 0.05 and ****P < 0.0001 indicate significance of differences from DMSO controls indicated. D: Comparison of effects of canagliflozin and empagliflozin, as in C. ****P < 0.0001. E: Comparison of effects of phloretin and phlorizin, as in C. ****P < 0.0001. Phosphorylation of AMPK and ACC in duplicate wells in response to canagliflozin (F) and dapagliflozin (G), from experiment in C; empagliflozin, from experiment in D (H); and phloretin (I) and phlorizin (J), from experiment in E. See Supplementary Fig. 1 for quantification of these blots.

like phenformin, canagliflozin inhibited the respiratory chain respiration in a concentration-dependent manner, whereas rather than the F1 ATP synthase. any effect on Complex II was marginal. The inhibitory ef- We next used primary mouse hepatocytes permeabilized fect of canagliflozin on Complex I was significantly greater with digitonin and measured the function of respiratory than that of dapagliflozin, with estimated half-maximal chain Complexes I and II. Figure 4D and E shows that can- effects at 18 6 1 mmol/L for canagliflozin and 40 6 agliflozin and dapagliflozin inhibited Complex I–supported 3 mmol/L for dapagliflozin. 2788 Canagliflozin Activates AMPK Diabetes Volume 65, September 2016

with a double knockout (DKO) of AMPK-a1 and -a2or WT controls. Figure 5A shows that AMPK was activated by concentrations of canagliflozin above 1 mmol/L in WT cells, associated with phosphorylation of AMPK and ACC (blots quantified in Supplementary Fig. 3A and B). As expected, AMPK activity was undetectable in DKO cells, and although ACC expression was normal, there was no basal or canagliflozin-stimulated ACC phosphorylation. We next measured lipid synthesis using [14C]acetate and fatty acid oxidation using [3H]palmitate (Fig. 5B and C). A769662 and phenformin, which activate AMPK by dif- ferent mechanisms, caused inhibition of lipid synthesis in the WT cells, whereas canagliflozin caused increasing in- hibition at 10 and 30 mmol/L. As expected, the inhibition by A769662 was abolished in DKO cells, but surprisingly, lipid synthesis was stimulated by both phenformin and canagliflozin in DKO cells. Fatty acid oxidation (Fig. 5C) was stimulated by AICAR, and this was abolished in the DKO cells, showing that it was AMPK-dependent. How- ever, phenformin and canagliflozin inhibited fatty acid oxidation in the WT and DKO cells. The likely explanation for this is that these drugs, although activating AMPK, inhibit the respiratory chain and, therefore, prevent reox- idation of NADH and FADH2 generated by fat oxidation, an effect that would be AMPK-independent and that has been previously observed in primary hepatocytes treated with metformin (22). Canagliflozin Inhibits Lipogenesis in Hepatocytes Through a Mechanism Involving Phosphorylation of ACC by AMPK The inhibition of lipid synthesis by canagliflozin in Fig. 5B was associated with phosphorylation of ACC at the AMPK site(s) (Fig. 5A; results quantified in Supplementary Fig. 3B). To test whether the effect of canagliflozin on lipid synthesis required phosphorylation of ACC1/ACC2, we studied hepatocytes from mice with a DKI of mutations at the AMPK sites on both ACC isoforms (22). Figure 5D shows that increasing concentrations of canagliflozin caused progressive inhibition of lipid synthesis in WT hepatocytes that was abolished, at least at concentrations of 10 mmol/L and below, in DKI cells. The effects of 500 mmol/L metformin were also abolished in the DKI cells. Figure 3—Comparison of AMPK activation by canagliflozin and However, at 30 mmol/L canagliflozin, there was still some phloretin in WT and RG cells. A: WT and RG HEK-293 cells were inhibition in the DKI cells, although significantly less than treated for 1 h with the indicated concentrations of canagliflozin, in WT cells. The effects of canagliflozin and metformin on and the results of kinase assays in anti-a immunoprecipitates are shown (mean 6 SEM, n = 4 to 6). ****P < 0.0001 indicates signif- lipid synthesis were accompanied by increased phosphory- icance of differences from vehicle control. B: Analysis of AMPK lation of AMPK and, in WT cells, of ACC. The pACC and phosphorylation in duplicate dishes of cells from the same experi- total ACC antibodies both detected doublets; the lower ment as in A. C: Experiment as in A, but using phloretin. ****P < D B and upper bands are ACC1 (predicted mass 265 kDa) and 0.0001. :Asin , but using phloretin. See Supplementary Fig. 2 for – quantification of these blots. ACC2 (276 kDa) (22). Figure 5E shows that 10 30 mmol/L canagliflozin significantly increased pT172/AMPK-a and pACC/ACC (ACC1 + ACC2) in WT mouse hepatocytes. AMPK-Dependent and -Independent Effects Canagliflozin Inhibits Glucose Uptake in HEK-293 Cells of Canagliflozin on Cellular Metabolism and MEFs To examine effects of canagliflozin on metabolic effects We next assessed the effects of the drugs on glucose mediated by AMPK, we initially used immortalized MEFs transport by measuring 2DG uptake. Canagliflozin inhibited diabetes.diabetesjournals.org Hawley and Associates 2789

Figure 4—Effects of canagliflozin, phloretin, and phenformin on cellular ADP-to-ATP ratios and oxygen uptake. A: Effects of phenformin and increasing concentrations of canagliflozin on cellular ADP-to-ATP ratios. HEK-293 cells were treated with the indicated drug concen- tration for 1 h, and cellular ADP-to-ATP ratios were determined by capillary electrophoresis of perchloric acid extracts (mean 6 SEM, n = 4). ***P < 0.001 and ****P < 0.0001 indicate significance of differences from vehicle control. B:AsinA, but using phloretin in place of canagliflozin. C: Effects of phenformin and increasing concentrations of canagliflozin on cellular oxygen uptake. At the point shown by the first arrow (“drug”), vehicle (DMSO), phenformin (10 mmol/L), or the indicated concentration of canagliflozin (mmol/L) was added, and oxygen uptake was measured at regular intervals. At the point shown by the second arrow (“DNP”), 2,4-DNP was added, and oxygen uptake was measured for a further 15 min. Results are expressed as percentage of the mean of the first three time points before drug addition (mean 6 SD; three replicate dishes performed twice, n = 6). After addition of drugs, the effects of 10 mmol/L canagliflozin were significantly different from the DMSO control by two-way ANOVA (Dunnett multiple comparison test) at all time points except 67.5 and 75 min; effects of 30 mmol/L canagliflozin and 10 mmol/L phenformin were significant at all time points. D and E: Complex I– and Complex II–supported respiration in permeabilized primary mouse hepatocytes. The cells were permeabilized with digitonin and then provided with ADP, glutamate, and malate to stimulate Complex I–supported respiration, or rotenone, succinate, and ADP to simulate Complex II–supported respiration. Results are expressed as percent inhibition of Complex I and II by canagliflozin and dapagliflozin (mean 6 SEM, n =3–5). ****P < 0.0001 indicates statistically significant differences between effects of canagliflozin and dapagliflozin by two-way ANOVA.

uptake by 50–60% in HEK-293 cells and MEFs, whereas although the band did not always comigrate with the the AMPK activator AICAR had no effect. The results were bands in control tissue, possibly due to variable glycosyl- identical in WT MEFs and AMPK DKO MEFs, showing ation (Fig. 6C). Attempts to measure expression of other that this effect of canagliflozin was AMPK-independent. glucose transporters (GLUT3, SGLT1) by Western blotting We also assessed the expression of SGLT2 in these cell were inconclusive. The effect of canagliflozin to inhibit types using Western blotting. HEK-293 cells and mouse glucose uptake in HEK-293 cells and MEFs appears to be liver, but not MEFs, expressed a polypeptide that comi- yet another off-target effect, because dapagliflozin had no grated with SGLT2 in mouse kidney. HEK-293 cells, effect on 2DG uptake in either cell type (Fig. 6A and B). To MEFs, and mouse liver also appeared to express GLUT1, further confirm that the activation of AMPK in HEK-293 2790 Canagliflozin Activates AMPK Diabetes Volume 65, September 2016

Figure 5—Effects of canagliflozin on AMPK, lipid synthesis, and fatty acid oxidation in intact cells. A: WT and AMPK-a12/2 and AMPK-a22/2 MEFs (AMPK-a1/a2 DKO) were incubated with the indicated concentrations of canagliflozin for 1 h, and AMPK activity was determined (upper panel) in anti–AMPK-a immunoprecipitates (mean 6 SEM, n = 4). Assays were performed in the DKO cells, although the activity, as expected, was negligible. *P < 0.05 and ****P < 0.0001 indicate significant differences from the vehicle control. The lower panel shows phosphorylation of AMPK and ACC analyzed in duplicate dishes of cells from the same experiment (see Supplementary Fig. 3 for quantification of these blots). B: Lipid synthesis (incorporation of radioactivity from [14C]acetate into total lipid, i.e., fatty acids and sterols) in WT and AMPK-a1/a2DKOMEFs incubated in the indicated concentrations of A769662, phenformin, or canagliflozinfor1h(mean6 SEM, n =4).**P < 0.01 and ****P < 0.0001 3 3 indicate significant difference from vehicle controls. C: Fatty acid oxidation (incorporation of radioactivity from [ H]palmitate into H2O) in WT and AMPK-a1/a2 DKO MEFs incubated in the indicated concentrations of phenformin, AICAR, or canagliflozin for 1 h. Results are mean 6 SEM, n = 4. ****P < 0.0001 indicates significant difference from vehicle controls. D: Lipid synthesis and protein phosphorylation in primary mouse hepatocytes from WT and ACC1/ACC2 DKI mice. The upper panel shows incorporation of radioactivity from [3H]acetate into total lipid measured after 4 h (mean 6 SEM, cells from three mice, each performed in triplicate). ****P < 0.0001 indicates significant differences from controlwithineachgenotype.†P < 0.05, ††P < 0.01, and ††††P < 0.0001 indicate significant difference between genotypes at the same drug concentration. The lower panel shows analysis of protein phosphorylation by Western blotting of a representative example. E: Quantification of the increases in phosphorylation in WT hepatocytes of Thr172 on AMPK (left) or Ser79 plus Ser212 on ACC1/ACC2 (right), normalized for the expression of total AMPK or ACC1/ACC2 and expressed relative to control (mean 6 SEM; n = 4 for pT172/AMPK-a,andn = 5 for pACC/ACC). *P < 0.05, **P < 0.01, and ****P < 0.0001 indicate significant differences from vehicle controls.

cells was not secondary to its effects on glucose uptake, we glucose caused a modest activation and Thr172 phosphor- compared the effects of canagliflozin with complete glucose ylation of AMPK, activation by 30 mmol/L canagliflozin removal from the medium (Fig. 6D;quantification of blots was more than threefold larger. Because inhibition of glu- in Supplementary Fig. 3C and D). Although removal of all cose uptake by canagliflozin was only partial (Fig. 6A), the diabetes.diabetesjournals.org Hawley and Associates 2791

Figure 6—Canagliflozin inhibits glucose transport in HEK-293 cells and MEFs, but this does not account for AMPK activation. A: Uptake of 2DG in HEK-293 cells treated with 10 mmol/L canagliflozin, 10 mmol/L dapagliflozin, or 1 mmol/L AICAR. Results are mean 6 SEM (n = 5). **P < 0.01 indicates significant differences from vehicle controls. B:AsinA, but in WT or AMPK-a12/2 and AMPK-a22/2 (AMPK-a1/a2 DKO) MEFs. C: Expression of SGLT2 and GLUT1; membrane fractions from MEFs or HEK-293 cells (80 mg protein), mouse kidney (KID) or liver (LIV) (30 mg), mouse brain (Br.) (15 mg), or human placenta (HUM PLA) (7.5 mg) were analyzed by Western blotting and probed using the indicated antibodies. D: Effect of total removal of medium glucose (center) or 30 mmol/L canagliflozin (right) on AMPK activity (top) and phosphorylation of AMPK (pT172) and pACC. Results in the bar chart are mean 6 SEM (n = 3). *P < 0.05, **P < 0.01, and ****P < 0.0001 indicate statistical significance of differences. effect of canagliflozin on AMPK is unlikely to be due to a was likely secondary to the reduction of blood glucose, which reduced supply of glucose for catabolism. was similar in WT and ACC DKI mice (Fig. 7D). Canagliflozin Activates AMPK in Mice In Vivo DISCUSSION To test whether canagliflozin activated AMPK in vivo, it Recent clinical trials suggest that the SGLT2 inhibitors was administered to mice (100 mg/kg) by oral gavage, and canagliflozin, dapagliflozin, and empagliflozin show promise tissues were collected 3 h later by freeze clamping in situ, for reversal of hyperglycemia, either as monotherapy or as which preserves the activation state of AMPK (35). In adjuncts to existing therapy. Compared with dapagliflozin, fi liver, Thr172 phosphorylation of AMPK was signi cantly canagliflozin also has consistently favorable effects on increased by this treatment, as was the phosphorylation of plasma lipid profiles (7,8,36,37). These differential effects ACC and Raptor at AMPK sites (Fig. 7A and Supplementary on plasma lipids prompted us to investigate whether cana- fi Fig. 4). By contrast, signi cant increases in phosphoryla- gliflozin might have SGLT2-independent effects. Our results tion of AMPK, ACC, and Raptor were not observed in show that canagliflozin causes a substantial activation of muscle (tibialis interior), gonadal white adipose tissue, or AMPK in human and mouse cells at concentrations corre- – spleen (Supplementary Fig. 5A C). We also measured the sponding to the peak plasma concentrations achieved after effects on the RER of oral administration of canagliflozin at therapeutic doses in humans. By contrast, dapagliflozin and the time of withdrawing food from previously fasted mice empagliflozin only caused a modest AMPK activation at that had been refed for 2 h. Canagliflozin caused a more concentrations well above their peak plasma concentrations. rapid drop in RER than vehicle in WT mice, indicating a Thus, activation of AMPK by dapagliflozin or empagliflozin more rapid shift back toward fat rather than carbohydrate is less likely to be significant in vivo. oxidation (Fig. 7B). However, this was also observed in Our results demonstrate that AMPK activation is pri- ACC1/ACC2 DKI mice, showing that the effect was indepen- marily due to inhibition of Complex I of the respiratory dent of ACC phosphorylation and, therefore, presumably chain, leading to increases in cellular AMP/ADP that bind of AMPK (Fig. 7C). This reduction in RER by canagliflozin to the g-subunit and promote Thr172 phosphorylation. 2792 Canagliflozin Activates AMPK Diabetes Volume 65, September 2016

Figure 7—Liver AMPK is activated by oral administration of canagliflozin in mice, but this does not explain changes in RER. A: Phos- phorylation of AMPK, ACC, and Raptor relative to total protein in mouse liver after oral administration of canagliflozin (100 mg/kg) to mice (mean 6 SEM, n =9–11). *P < 0.05 indicates significant difference by Student t test. B: RER (VCO2/VO2) in 12-h fasted mice that had been refed a carbohydrate-rich diet for 2 h between 7:00 and 9:00 A.M. and were then administered vehicle or canagliflozin (30 mg/kg) at the time that food was withdrawn again. Results are mean 6 SEM (n =10–12). C:AsinB, but experiments performed in ACC1/ACC2 DKI mice. Results are mean 6 SEM (n =5–7). D: Glucose concentrations in the tail vein during experiments of the type shown in B and C.*P < 0.05 and **P < 0.01 indicate statistically significant differences in canagliflozin samples vs. vehicle control. hr, h.

Thus, canagliflozin: inhibited 2DG uptake in HEK-293 cells and MEFs in an AMPK-independent manner, indicating that it had addi- 1. increased cellular ADP-to-ATP ratios; tional off-target effects on glucose transport, presumably 2. increased AMPK activation and Thr172 phosphoryla- due to inhibition of another glucose transporter such as tion in cells expressing the WT but not the AMP/ GLUT1. Indeed, previous studies in L6 myotubes have in- ADP-insensitive R531G mutant of AMPK-g2; dicated that 10 mmol/L canagliflozin can inhibit glucose 3. inhibited oxygen uptake in HEK-293 cells; and uptake by ;50%, an effect that was attributed to GLUT1 4. inhibited oxygen uptake in permeabilized mouse hepato- inhibition (38). However, this is unlikely to account for the cytes provided with substrates that feed into Complex I. AMPK activation observed in our experiments, because even complete removal of glucose from the medium had only a Dapagliflozin also caused a less potent effect on Complex modest effect on AMPK activity compared with canagliflozin. I, although only at concentrations (10–30 mmol/L) higher Interestingly, we found that phloretin, the aglycone than those observed in human plasma with normal doses. derivative of phlorizin, also activated AMPK, although We also found that canagliflozin, but not dapagliflozin, phlorizin itself was much less effective. Like canagliflozin, diabetes.diabetesjournals.org Hawley and Associates 2793 phloretin appeared to act by increasing cellular AMP. observed in the DKI mice and was, therefore, presumably Phlorizin (39) and phloretin (40) were both previously independent of AMPK. It is possible that a reduction in reported to inhibit the function of isolated mitochondria, blood glucose caused by canagliflozin causes increased but we found that only phloretin is effective in intact cells, fat oxidation due to competition between glucose and perhaps due to greater membrane permeability. fat for substrate oxidation (42) and that this obscures Our studies also show that AMPK activation has the any effect due to ACC phosphorylation by AMPK. This expected effects on lipid synthesis in intact cells. Thus, might be addressed in future studies using SGLT2 knock- three distinct AMPK activators—A769662, phenformin, out mice. and canagliflozin—all inhibited the pathway in MEFs, and Our findings raise the interesting question of whether these inhibitory effects were abolished in MEFs lacking dual therapy with canagliflozin and metformin would be AMPK, correlating with a complete loss of ACC phosphor- more effective than canagliflozin alone. In a recently re- ylation. Surprisingly, phenformin and canagliflozin (but ported clinical trial of newly diagnosed subjects with type 2 not A769662) stimulated lipid synthesis in DKO cells. diabetes (43), monotherapy with canagliflozin was more When we measured fatty acid oxidation in the same cells, effective in lowering HbA1c than metformin. Although phenformin and canagliflozin inhibited the pathway in an dual therapy was more efficacious than canagliflozin alone, AMPK-independent manner, which is expected because the effects of the two drugs were not additive, as might be both compounds inhibit Complex I. The major fates of expected if they had distinct mechanisms of action. By cellular acetyl-CoA are oxidation by the tricarboxylic contrast, in a similar trial using metformin and dapagliflo- acid cycle or incorporation into lipids. Thus, in the ab- zin, monotherapy with dapagliflozin was not more effective sence of AMPK to inhibit lipid synthesis, acetate flux than metformin, and the effects of dual therapy were closer might be diverted into lipid synthesis as oxidation was to being additive (44). inhibited. This provides an explanation for the paradoxi- Finally, although the long-term effects of metformin to cal activation of lipid synthesis by phenformin and cana- inhibit hepatic glucose production in mice are AMPK- gliflozin observed in AMPK knockout cells. dependent (22), studies have suggested that its rapid ef- Our results in isolated mouse hepatocytes demonstrate fects are AMPK-independent (20,21). However, the latter that the effects of canagliflozin on lipid synthesis are authors agree that the primary effect of metformin is to mediated by phosphorylation of ACC. Thus, a moderate inhibit the respiratory chain and increase cellular AMP in and therapeutically relevant concentration of canagliflo- the liver, which is then proposed to affect other AMP- zin (10 mmol/L) inhibited lipid synthesis in WT hepato- sensitive targets such as fructose-1,6-bisphosphatase (20) cytes but failed to do so in DKI cells from mice where both or adenylate cyclase (21). Because canagliflozin inhibits the ACC isoforms lacked the critical AMPK site. At higher respiratory chain and activates AMPK in the liver, it is con- canagliflozin (30 mmol/L), there was some inhibition ceivable that these AMP-dependent but AMPK-independent even in DKI hepatocytes, although significantly less effects of metformin might also be mimicked by canagliflozin. than in WT cells; this is most likely because both ACC isoforms use ATP as a direct substrate. Once the increase in the cellular ADP-to-ATP ratio becomes substantial, as Acknowledgments. The authors thank Benoit Viollet (Institut Cochin, with 30 mmol/L canagliflozin (Fig. 4A), decreases in ATP Paris) for AMPK knockout MEFs. may limit flux through ACC independently of AMPK. This Funding. S.J.M. and I.P.S. were supported by a Project Grant (PG/13/82/30483) is a revealing demonstration of the physiological role of from the British Heart Foundation. The study was supported by the Canadian AMPK: as ATP falls under situations of energetic stress, Diabetes Association (G.R.S.), by the Canadian Institutes of Health Research (G.R.S.), the kinase limits the function of energy-consuming path- by a Senior Investigator Award from the Wellcome Trust (097726) to D.G.H., and by ways before the ATP concentration has dropped to levels the pharmaceutical companies supporting the Division of Signal Transduction where it becomes limiting for the pathway itself. This Therapy at Dundee (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Merck KGaA, Janssen Pharmaceuticals, and Pfizer). G.R.S. is a Canada Research Chair in occurs because AMPK is more sensitive to ATP depletion Metabolism and Obesity and the J. Bruce Duncan Chair in Metabolic Diseases. than the ATP-consuming enzymes in the pathway. Duality of Interest. No potential conflicts of interest relevant to this article Our results show that oral administration of canagli- were reported. flozin increased Thr172 phosphorylation of AMPK in liver Author Contributions. S.A.H., R.J.F., B.K.S., G.J.G., S.J.M., R.D.P., and in vivo and phosphorylation of ACC and Raptor, two of its E.A.D. established experimental protocols and/or conducted experiments. I.P.S., well-recognized downstream targets. Although we only G.R.S., and D.G.H. supervised research. G.R.S. and D.G.H. wrote the initial tested effects of the drug in normal mice, there is no manuscript. All authors contributed revisions and corrections. G.R.S. and D.G.H. reason to believe the results would be any different in are the guarantors of this work and, as such, had full access to all the data in the diabetic models; for example, berberine, which activates study and take responsibility for the integrity of the data and the accuracy of the AMPK by the same mechanism as canagliflozin (19), ac- data analysis. tivates AMPK normally in db/db mice (41). Canagliflozin References also caused a more rapid drop in the RER when admin- 1. Rosenwasser RF, Sultan S, Sutton D, Choksi R, Epstein BJ. SGLT-2 in- istered to fed mice, indicating a more rapid switch to fat hibitors and their potential in the treatment of diabetes. Diabetes Metab Syndr versus carbohydrate oxidation. However, this was still Obes 2013;6:453–467 2794 Canagliflozin Activates AMPK Diabetes Volume 65, September 2016

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