bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 TITLE 2 Compound- and fiber type-selective requirement of AMPK3 for insulin-independent 3 glucose uptake in skeletal muscle 4 5 AUTHORS 6 Philipp Rhein1,2, Eric M. Desjardins3,4†, Ping Rong5†, Danial Ahwazi6, Nicolas Bonhoure1, Jens Stolte1, 7 Matthieu D. Santos7, Ashley J. Ovens8,9, Amy M. Ehrlich6, José L. Sanchez Garcia1, Qian Ouyang5, 8 Mads F. Kjolby10, Mathieu Membrez1, Niels Jessen10, Jonathan S. Oakhill8,9, Jonas T. Treebak6, 9 Pascal Maire7, John W. Scott9,11,12, Matthew J. Sanders1, Patrick Descombes1, Shuai Chen5, Gregory 10 R. Steinberg3,4, Kei Sakamoto1,6* 11 12 AFFILIATIONS 13 1Nestlé Research, Société des Produits Nestlé S.A., EPFL Innovation Park, Lausanne, 1015, 14 Switzerland.

15 2School of Life Sciences, EPFL Innovation Park, Lausanne, 1015, Switzerland.

16 3Centre for Metabolism, Obesity, and Diabetes Research, McMaster University, Hamilton, ON, 17 L8N3Z5, Canada

18 4Department of Medicine and Department of Biochemistry and Biomedical Sciences, McMaster 19 University, Hamilton, ON, L8N3Z5, Canada

20 5MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, School 21 of Medicine, Nanjing University, Nanjing, 210061, China

22 6Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, 23 Copenhagen, 2200, Denmark

24 7Université de Paris, Institut Cochin, INSERM, CNRS. 75014 Paris, France.

25 8Metabolic Signalling Laboratory, St Vincent’s Institute of Medical Research, School of Medicine, 26 University of Melbourne, Fitzroy, VIC 3065, Australia.

27 9Mary MacKillop Institute for Health Research, Australian Catholic University, Fitzroy, VIC 3000, 28 Australia.

29 10Department of Biomedicine, Aarhus University, Aarhus Denmark, Department of Clinical 30 Pharmacology and Steno Diabetes Center Aarhus, Aarhus University Hospital, Aarhus, Denmark.

31 11Protein Chemistry and Metabolism Unit, St Vincent’s Institute of Medical Research, Fitzroy, VIC 32 3065, Australia. bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

33 12The Florey Institute of Neuroscience and Mental Health, Parkville, VIC 3052, Australia

34 AUTHOR LIST FOOTNOTES 35 †These authors contributed equally.

36 *Corresponding author

37

38 CORRESPONDING AUTHOR CONTACT INFORMATION

39 Kei Sakamoto

40 Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, 41 Blegdamsvej 3B, Copenhagen DK-2200, Denmark. E-mail: [email protected] 42 Phone: (+45) 9356 3553 43 44

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45 ABSTRACT 46 Objective: The metabolic master-switch AMP-activated protein kinase (AMPK) mediates insulin- 47 independent glucose uptake in muscle and regulates the metabolic activity of brown and beige 48 adipose tissue (BAT). The regulatory AMPK3 isoform is uniquely expressed in skeletal muscle 49 and also potentially in BAT. Here, we investigated the role that AMPK3 plays in mediating skeletal 50 muscle glucose uptake and whole-body glucose clearance in response to small-molecule 51 activators that act on AMPK via distinct mechanisms. We also assessed if 3 plays a role in 52 adipose thermogenesis and browning.

53 Methods: Global AMPK3 knockout (KO) mice were generated. A systematic whole-body, tissue 54 and molecular phenotyping linked to glucose homeostasis was performed in 3 KO and wild type 55 (WT) mice. Glucose uptake in glycolytic and oxidative skeletal muscle ex vivo, as well as blood 56 glucose clearance in response to small molecule AMPK activators that target nucleotide-binding 57 domain of  subunit (AICAR) and allosteric drug and metabolite (ADaM) site located at the 58 interface of the  and  subunit (991, MK-8722) were assessed. Oxygen consumption, 59 thermography, and molecular phenotyping with a β3-adrenergic receptor agonist (CL-316,243) 60 treatment were performed to assess BAT thermogenesis, characteristics and function.

61 Results: Genetic ablation of 3 did not affect body weight, body composition, physical activity, and 62 parameters associated with glucose homeostasis under chow or high fat diet. 3 deficiency had no 63 effect on fiber-type composition, mitochondrial content and components, or insulin-stimulated 64 glucose uptake in skeletal muscle. Glycolytic muscles in 3 KO mice showed a partial loss of 65 AMPK2 activity, which was associated with reduced levels of AMPK2 and 2 subunit isoforms. 66 Notably, 3 deficiency resulted in a selective loss of AICAR-, but not MK-8722-induced blood 67 glucose-lowering in vivo and glucose uptake specifically in glycolytic muscle ex vivo. We detected 68 3 in BAT and found that it preferentially interacts with 2 and 2. We observed no differences in 69 oxygen consumption, thermogenesis, morphology of BAT and inguinal white adipose tissue 70 (iWAT), or markers of BAT activity between WT and 3 KO mice.

71 Conclusions: 72 These results demonstrate that 3 plays a key role in mediating AICAR- but not ADaM site binding 73 drug-stimulated blood glucose clearance and glucose uptake specifically in glycolytic skeletal 74 muscle. We also showed that 3 is dispensable for thermogenesis and browning of iWAT.

75 KEY WORDS 76 AMP-activated protein kinase; 5-aminoimidazole-4-carboxamide riboside; MK-8722; glucose 77 uptake; TBC1D1; Brown adipose tissue

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78 1. INTRODUCTION 79 AMP-activated protein kinase (AMPK) is an evolutionary conserved energy sensor that functions to 80 maintain energy homeostasis through coordinating metabolic pathways [1; 2]. AMPK exists as 81 complexes of three subunits; a catalytic α and two regulatory β and γ subunits. Each exists as 82 multiple isoforms (α1/α2, β1/β2, γ1/γ2/γ3), generating up to twelve possible combinations [1]. 83 AMPK heterotrimers are active when a conserved threonine (Thr172) residue within the activation 84 loop of the  subunit kinase domain is phosphorylated [3]. The major upstream kinase 85 phosphorylating Thr172 in metabolic tissues (e.g., muscle, liver) is a complex containing LKB1 [4; 86 5]. The -subunits contain four tandem cystathionine -synthase (CBS) motifs that bind adenine 87 nucleotides. Binding of ADP and/or AMP to CBS motifs causes conformational changes that 88 promote net Thr172 phosphorylation [6-8]. Moreover, the binding of AMP, but not ADP, further 89 increases AMPK activity by direct allosteric stimulation [6]. Prodrugs of AMP-mimetics such as 5- 90 aminoimidazole-4-carboxamide riboside (AICAR) have been widely used as pharmacological 91 AMPK activators that target the CBS motifs [9]. Proof of concept preclinical studies demonstrated 92 that AICAR treatment improved insulin sensitivity in animal models of insulin resistance [10]. 93 However, AICAR produces numerous AMPK-independent metabolic actions [11]. For example, we 94 have recently demonstrated that AICAR suppresses hepatic glucose production independently of 95 AMPK [12] through inhibition of fructose-1,6-bisphosphatase-1, an AMP-sensitive enzyme involved 96 in gluconeogenesis, in vivo [13]. We also showed that AICAR regulated >750 genes in AMPK-null 97 mouse primary hepatocytes [14]. 98 A nucleotide-independent regulation of AMPK was discovered when a novel small- 99 molecule activator, A-769662, was identified [15] and mechanism of action explored [16-18]. The 100 crystallographic structures of AMPK trimeric complexes revealed that A-769662 and 991 (another 101 activator, also known as ex229) bind in a pocket termed allosteric drug and metabolite (ADaM) site 102 located at the interface of the α subunit (kinase domain N-lobe) and  subunit (carbohydrate 103 binding module) [9; 19; 20]. A-769662 was subsequently found to be selective for the AMPK1- 104 containing complexes [17] and failed to stimulate AMPK-dependent glucose uptake due to lack of 105 potency against 2-containing complexes that are prevalent in skeletal muscle [21]. We and others 106 have shown that 991, and its two related benzimidazole derivatives with improved bioavailability 107 (MK-8722, PF-739), are potent and highly-specific AMPK activators [14; 22; 23]. They activate 108 both 1- and 2-containing complexes (thereby activating all twelve possible human AMPK 109 complexes) and have been shown to stimulate glucose uptake in skeletal muscle and lower blood 110 glucose levels in vivo [22; 24]. Notably, administration of PF-739 resulted in attenuated blood 111 glucose reduction in skeletal muscle-specific but not in liver-specific double knockout of 112 AMPKα1/α2 [23].

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113 AMPK isoform expression varies among different cell and tissue types, with 1, 1, and 1 114 appearing the most ubiquitously expressed. Conversely, 3 is selectively expressed in skeletal 115 muscles containing a high proportion of glycolytic/fast-twitch fibers such as extensor digitorum 116 longus (EDL) muscle [22; 25-27]. Interestingly, even though skeletal muscle expresses multiple 117 isoforms, assays of immunoprecipitated isoforms reveal the α2β2γ1 and α2β2γ3 complexes 118 account for 90% (of which α2β2γ3 accounts for 20%) of the total AMPK trimers in mouse EDL 119 skeletal muscle [21]. Loss of expression/function of 2, 2 or 3 is sufficient to ablate AICAR- 120 induced glucose uptake in isolated skeletal muscle ex vivo [25; 28-32]. 121 In addition to its established metabolic roles in skeletal muscle [33; 34], AMPK also plays a 122 vital role in regulating the development of brown adipose tissue (BAT), maintenance of BAT 123 mitochondrial function, and browning of white adipose tissue (WAT) [35]. Adipose-specific 124 AMPK1/2-null (ad-AMPK KO) mice had a profound defect in thermogenesis [36], and both cold 125 exposure and acute treatment with the β3-adrenergic receptor agonist (CL-316,243) in the ad- 126 AMPK KO mice yielded subnormal increments in oxygen consumption and BAT temperature 127 responses (likely related to impairments in BAT mitochondrial function). A high-throughput screen 128 of protein kinases using a combination of RNAi-mediated knockdown and pharmacological 129 inhibitors identified AMPK as a prominent kinase that promoted the formation of UCP1-abundant 130 brown adipocytes in vitro [37]. Proof of concept experiments in vivo demonstrate that daily 131 treatment of diabetic ZDF rats with an AMPK activator (C163, for 6 weeks) increased the formation 132 of brown adipocytes [37]. Intriguingly, transcripts of the Prakg3 (AMPK3 gene) were identified in 133 brown adipocyte precursors at intermediate levels, and RNAi-mediated knockdown of Prakg3 was 134 sufficient to profoundly block the brown adipocyte formation without affecting general adipose 135 differentiation [37]. These results prompted us to assess if 3 plays a role in adipose 136 thermogenesis and browning in vivo. 137 We hypothesized that 3-containing complexes play an important role for insulin- 138 independent and AMPK activator-mediated glucose uptake in skeletal muscle and for regulating 139 BAT thermogenesis. To test this hypothesis, we generated 3 KO mice and determined the effect 140 of AICAR and the ADaM site binding drugs (991, MK-8722) on glucose uptake in glycolytic and 141 oxidative skeletal muscles ex vivo and also blood glucose kinetics in vivo. In addition, we probed 142 BAT function using the 3-AR agonist CL-316,243. Strikingly, we found that 3 deficiency resulted 143 in a selective loss of AICAR-, but not 991/MK-8722-induced blood glucose clearance in vivo and 144 glucose uptake specifically in glycolytic muscle ex vivo. We also found that 3 is not required for 145 the acute induction of UCP1-mediated non-shivering thermogenesis in the BAT, for the adaptive 146 response to non-shivering thermogenesis or the browning of WAT. 147

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148 2. MATERIALS AND MTHODS 149 2.1. Materials 150 5-aminoimidazole-4-carboxamide riboside (AICAR) was purchased from Apollo Scientific 151 (OR1170T; Bredbury, United Kingdom). 991 (5-[[6-chloro-5-(1-methylindol-5-yl)-1H-benzimidazol- 152 2-yl]oxy]-2-methyl-benzoic acid) (CAS#: 129739-36-2) was synthesized by Spirochem (Basel, 153 Switzerland) as previously described [1]. Protein G Sepharose and P81 paper were purchased 154 from GE Healthcare (Chicago, IL, USA). [-32P]-ATP was purchased from PerkinElmer (Waltham, 155 MA, USA). The substrate peptide AMARA was synthesized by GL Biochem (Shanghai, China). All 156 other reagents were from MilliporeSigma (Burlington, MA, USA) if not otherwise stated. Lists of 157 primary and secondary antibodies are in the Supplementary Table 1 and 2. 158 159 2.2. Animal ethics and models 160 Animal experiments were approved by the internal and local ethics committee and conducted in 161 accordance with the European Convention for the Protection of Vertebrate Animals used for 162 Experimental and Other Scientific Purposes. Protocols used were approved by the Service 163 Vétérinaire Cantonal (Lausanne, Switzerland) under licenses VD3332 and VD3465, and also 164 approved by an ethical committee (Com’Eth, CE17) registered at the French Ministry of Research 165 under the reference: 10261, as well as were in accordance with McMaster Animal Care Committee 166 guidelines (AUP #: 16-12-41, Hamilton, ON), and the Ethics Committees at Nanjing University with 167 involved personnel having personal licenses from the regional authority. The generation of a 168 constitutive Prkag3−/− (AMPKγ3−/−) mice was performed by Taconic Biosciences as described in 169 Supplementary Figure 1. The AMPK1f/f and AMPK2f/f mice were as previously described [38], 170 and obtained from the Jackson Laboratory (Bar Harbor, ME, USA). These two strains were used to 171 derive AMPK1f/f/2f/f mice that were then bred with the Mlc1f-Cre mice to obtain the 172 AMPK1f/f/2f/f - Mlc1f-Cre mice. The resultant AMPK1f/f/2f/f - Mlc1f-Cre mice are the 173 AMPK1/2 skeletal muscle-specific knockout mice. TBC1D1 S231A knock-in mice have been 174 described [39]. All these lines are on C57BL6 background. The animals were kept and maintained 175 according to local regulations under a light-dark cycle of 12 hours and had free access to a 176 standard chow diet. Male mice ranging 10-16 weeks of age were used for experiments otherwise 177 stated. High fat diet (60% kcal% fat) was obtained from Research Diet (RD 12492). 178 179 2.3. Analysis of body composition and plasma hormone levels 180 Body composition (fat content, lean tissues and free body fluid) was assessed using the Minispec 181 analyser (Bruker) by Nuclear Magnetic Resonance (NMR) technology. The test was conducted on 182 conscious fed mice. Blood was collected at the indicated age by retro orbital puncture under

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183 isoflurane anesthesia at noon on mice fasted for 4 hours. Plasma insulin and leptin levels were 184 measured on a BioPlex analyser (BioRad) using the Mouse Metabolic Magnetic Hormone 185 Magnetic Bead panel kit (MilliporeSigma). 186 187 2.4. Oral glucose tolerance test 188 Mice were fasted overnight (16 hours) and a bolus of glucose solution (2 g/kg body weight) was 189 administered via oral gavage. Blood glucose collected from tail vein was measured at different time 190 points over 120 min using blood glucose monitor and glucose test strips (Roche Diagnostics, Accu- 191 Chek). 192 193 2.5. Preparation of mouse tissue extracts for protein analysis 194 Mouse tissue were dissected and immediately frozen in liquid nitrogen. The tissues were 195 homogenized in ice-cold lysis buffer (270 mM sucrose, 50 mM Tris·HCl (pH 7.5), 1 mM EDTA, 1

196 mM EGTA, 1% (w/v) Triton X-100, 20 mM glycerol-2-phosphate, 50 mM NaF, 5 mM Na4P2O7, 1 197 mM DTT, 0.1 mM PMSF, 1 mM benzamidine, 1 μg/ml microcystin-LR, 2 g/ml leupeptin, and 2 198 g/ml pepstatin A) using a tissue lyser (Tissue Lyser II; Qiagen). Lysates were centrifuged at 199 21,300 g for 15 min and protein concentration from the supernatant was determined using 200 Bradford reagent (23200, ThermoFisher) and bovine serum albumin (BSA) as standard. The 201 supernatants were stored in aliquots in -80°C freezer until subsequent analysis. 202 203 2.6. Immunoblotting 204 Protein extracts were denatured in Laemmli buffer at 95°C for 5 min. 20 g of protein was 205 separated by SDS-PAGE on 4-12% gradient gels (NW04127, ThermoFisher) and transferred onto 206 nitrocellulose membranes (#926-31090, LiCOR). Membranes were blocked for 1 hour at room 207 temperature in LiCOR blocking buffer (#927-60001, LiCOR). The membranes were subsequently 208 incubated in TBST (10 mM Tris (pH 7.6), 137 mM NaCl, and 0.1% (v/v) Tween-20) containing 5% 209 (w/v) BSA and the primary antibody overnight at 4°C. After extensive washing, the membranes 210 were incubated for 1 hour in either HRP-conjugated or LiCOR secondary antibodies diluted 211 1:10,000. Signal imaging was performed either using enhanced chemiluminescence (ECL) reagent 212 (GE Healthcare) or a LiCOR Odyssey CLx imaging system. Densitometry for ECL blots was 213 performed using Image J Software (NIH). Due to sample limitation (from the incubated muscle 214 tissue samples), we also utilized automated capillary Western Blot system Sally Sue 215 (ProteinSimple, San Jose, CA, USA). Experiments were performed according to the 216 manufacturer’s protocol using the indicated standard reagents for the Sally Sue system (SM-S001, 217 ProteinSimple). Briefly, all samples were first diluted to 2 mg/ml in lysis buffer and then further

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218 diluted to 0.5 mg/ml in 0.1% SDS. Following the manufacturer’s instructions for sample 219 preparation, this resulted in an assay protein concentration of 0.4 mg/ml. 220 221 2.7. Immunoprecipitation and in vitro AMPK activity assay 222 Lysates of muscle (200 g) or BAT (1,000 g) were incubated on a rotating platform at 4°C 223 overnight with a mix of 5 μl protein G-sepharose and the indicated antibodies. The beads were 224 pelleted at 500 g for 1 min and initially washed twice with 0.5 mL lysis buffer containing 150 mM 225 NaCl and 1 mM DTT and subsequently washed twice with the same amount of buffer A [50 mM 226 HEPES (pH 7.4), 150 mM NaCl, 1 mM EGTA and 1 mM DTT]. The AMPK complexes were either 227 eluted with Laemmli buffer for immunoblot analysis or taken directly for AMPK activity 228 measurement. The AMPK activity assay was performed by incubating the beads (immune- 229 complexes) for 30 min at 30°C on a heated shaker in buffer A with additional 10 mM Mg2+ and 100 230 M ATP in presence of 200 M AMARA peptide (AMARAASAAALARRR) and 1 Ci of [-32P] ATP 231 [22]. Reactions were stopped by spotting the reaction mix onto P81 filter paper and washing in 75 232 mM phosphoric acid. The P81 papers were dried after three washes and the 32P incorporation into 233 the substrate peptide measured by Cherenkov counting (5 min) using a scintillation counter (Tri- 234 Carb 2810TR, PerkinElmer). 235 236 2.8. Citrate synthase activity assay 237 Protein extracts (10 µg) (using the same lysis buffer described above) was assayed in duplicates 238 using a citrate synthase assay kit (CS0720, MilliporeSigma) according to the manufacturer’s 239 instruction using recombinant citrate synthase as positive control. 240 241 2.9. Analysis of gene expression and mitochondrial DNA copy number using quantitative real-time 242 PCR (qPCR) 243 To perform a relative quantification of mRNA levels of the AMPK subunit isoforms in mouse 244 skeletal muscle tissues, reverse transcription and qPCR was performed as described [14]. All the 245 primers and sequences are listed in the Supplementary Table 3. Relative mRNA quantities were 246 calculated for triplicate muscle samples from 4-5 animals and normalized using the three reference 247 genes Hprt1 (hypoxanthine ribosyltransferase, HPRT), GusB (beta-glucuronidase) and Pgk1 248 (Phosphoglycerate Kinase 1). Real-time qPCR in brown adipose tissue was performed separately 249 as described [36]. Relative gene expression was calculated using the comparative Ct (2-ΔCt) 250 method, where values were normalized to a reference gene (Ppia). 251 To relatively quantify the amount of mtDNA present per nuclear genome by qPCR, mtDNA (16S, 252 ND4) and nuclear DNA (PMP22, Titin) primers and probes were used, the sequences of which are

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253 shown in Supplementary Table 3. The relative mt copy number was determined based on the 254 relative abundance of nuclear and mtDNA, calculated as average of the two targets respectively. 255 The relative abundance is then expressed by ΔCT or CT(nDNA) - CT(mtDNA) and displayed as 256 fold change of copy number of mtDNA per nuclear genome compared to the WT muscle. 257 258 2.10. Immunofluorescence for fiber type determination and fiber size 259 For immunostaining against Myh4, Myh2, Myh7 and Laminin, mouse hindlimbs (no skin) without 260 fixation were embedded with Tissue-TEK OCT (Sakura Finetek, Netherlands) and directly frozen in 261 cold isopentane pre-cooled in liquid nitrogen as described [40]. Hindlimb cross sections were 262 prepares using a cryostat (Leica 3050s) with a thickness of 10 m. The cross sections were 263 washed 3 times for 5 min with PBS and then incubated with blocking solution (PBS and 10% goat 264 serum) for 30 min at room temperature. Sections were incubated overnight with primary antibody 265 diluted in PBS + 10% goat serum solution at 4°C and washed as described above. The sections 266 were incubated with secondary antibody, diluted in PBS + 10% goat serum solution for 1 hour at 267 room temperature. Sections were further washed and mounted with Mowiol solution and a glass 268 coverslip. Images were collected with a microscope (Olympus BX63F) and a camera (Hamamatsu 269 ORCA-Flash 4.0). Images were analyzed with ImageJ (NIH). Fiber boundaries were defined by the 270 laminin signal and myosin Myh7 (type I), Myh2 (type IIA) and Myh4 (type IIB) heavy chains were 271 quantified. Remaining unlabeled fibers were included for total fiber number and individual 272 proportions of type I, type IIA and type IIB of that total number calculated. 273 274 2.11. Ex vivo skeletal muscle incubation and analysis of glucose uptake 275 Animals were anesthetized by Avertin [2,2,2-Tribromoehtanol (Sigma-Aldrich #T48402) and 2- 276 Methyl-2-butanol 99% (Sigma-Aldrich #152463)] via intraperitoneal injection, and EDL or soleus

277 muscles were rapidly dissected and mounted in oxygenated (95% O2 and 5% CO2), and warmed 278 (30°C) Krebs-Ringer buffer in a myograph system (820MS DMT, Denmark). The respective 279 muscles were incubated as described [22; 41] in the presence of the indicated drug or vehicle for 280 50 min. 2-deoxy-[3H] glucose uptake was measured during the last 10 min of the incubation as 281 described [22; 39]. 282 283 2.12. AICAR and MK-8722 tolerance test 284 Access of the mice to food was restricted for 3 hours (07:00-10:00) prior to the experiment. AICAR 285 (250 mg/kg body weight) or vehicle (water) was injected intraperitoneally and blood glucose levels 286 were monitored for 120 min using the Contour XT glucometer (Bayer, Leverkusen) and single use 287 glucose sensors (Ascensia, Basel). MK-8722 tolerance was tested by oral administration of either

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288 MK-8722 (10 or 30 mg/kg body weight) or vehicle (0.25% (w/v) methylcellulose, 5% (v/v) 289 Polysorbate 80, and 0.02% (w/v) sodium lauryl sulfate in deionized water) [24]. Blood glucose 290 measurement was performed as described above for AICAR. 291 292 2.13. ZMP and adenine nucleotide measurements 293 Muscle tissues were lysed in 200 l cold 0.5 M perchloric acid. Extracts were collected and 294 clarified at 14,000 rpm for 3 min. 100 l clarified lysate was neutralized with 25 l cold 2.3 M

295 KHCO3 and incubated on ice for 5 min. Samples were centrifuged at 14,000 rpm for 3 min. ZMP 296 were measured by LC-MS/MS with modifications to our previously described method [42]. Both LC 297 and MS instruments were controlled and managed with the Analyst 1.7.1 software (AB Sciex). The 298 autosampler was set at 4°C and column oven set at 30°C, which housed a 150 mm (length) x 0.5 299 mm (inner diameter) Hypercarb 3 m porous graphitic carbon column (Thermo Fisher Scientific). 300 The LC solvent system comprised of 50 mM triethylammonium bicarbonate buffer (TEAB, 301 MilliporeSigma) pH 8.5 in pump A, and acetonitrile with 0.5 % trifluoroacetic acid (TFA; Sigma- 302 Aldrich) in pump B. A flow rate of 400 l/min was used throughout a gradient program consisting of 303 0 % B (2 min), 0 to 100 % B (10 min), 100 % B (3 min), 0 % B (2 min). Data was analyzed with 304 MultiQuant 3.0.2 software (AB Sciex) using area under the LC curve. Calibration curves were 305 determined by linear regression of the peak area of a ZMP standard curve and were required to 306 have a correlation coefficient (R2) of > 0.98. Adenine nucleotides and adenylate energy charge 307 were measured by LC-MS as described [43]. 308 309 2.14. Adipose tissue histology 310 Tissues were fixed in 10% formalin for 24-48 hours at 4°C and processed for paraffin embedding 311 and hematoxylin and eosin staining by the core histology laboratory at the McMaster Immunology 312 Research Centre (Hamilton, Canada). 313 314 2.15. Infrared thermography 315 UCP1-mediated thermogenesis was assessed in 14-week-old male wild-type and 3 KO mice as 316 described [44]. Briefly, mice were anaesthetized with an intraperitoneal injection of 0.5 mg/g body 317 weight Avertin (2,2,2-Tribromoethanol dissolved in 2-methyl-2-butanol; MilliporeSigma) then, 2 min 318 later, injected with either saline or the highly selective β3-adrenergic receptor agonist CL 316,243 319 (Tocris, Bristol, United Kingdom). Mice were subsequently placed dorsal side up onto an enclosed

320 stationary treadmill to measure oxygen consumption (VO2) with a Comprehensive Laboratory 321 Animal Monitoring System (Columbus Instruments, OH, USA) and 18 min after the second 322 injection a static dorsal thermal image was taken with an infrared camera (FLiR Systems,

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323 Wilsonville, OR, USA). Serum samples were collected via tail-nick just after the infrared image was 324 taken and non-esterified free fatty acid (NEFA) concentration was determined using manufacturer 325 instructions with a two-step kit (NEFA-HR 2, WAKO). 326 327 2.16. Metabolic monitoring 328 Metabolic monitoring was performed as described [45] in a Comprehensive Laboratory Animal 329 Monitoring System. For the chronic 5-day CL 316,243 challenge, mice were injected 330 intraperitoneally with saline or CL 316,243 (first 4 days 0.5 mg/kg and last day 1.0 mg/kg) at 09:30

331 hours and measurements for VO2 were calculated 6 hours post-injection. Mice were euthanized 24 332 hours after the last injection. 333 334 2.17. Statistical analysis 335 Data are reported as mean ± SEM and statistical analysis was performed using GraphPad Prism 336 software. As indicated in the respective figure legends, differences between only two groups were 337 analyzed using an unpaired two-tailed Student’s t-test and for multiple comparisons one-way 338 analysis of variance (ANOVA) with Bonferroni post hoc test or repeated measures two-way 339 ANOVA was used. Statistical significance was accepted at P < 0.05. 340

341 3. RESULTS 342 3.1. A genetic/constitutive loss of the AMPK3 reduced AMPK2 and 2 protein abundance 343 in mouse glycolytic skeletal muscle. 344 We generated constitutive AMPK3 KO mice through flanking exons 5-10 of Prkag3 gene with 345 LoxP sites (Supplementary Fig. 1A). This is expected to cause a loss-of-function of the Prkag3 346 gene by deleting the nucleotide binding cystathionine β-synthase (CBS)-2 domain and parts of the 347 CBS-1 and -3 domains and by generating a frame shift from exon 4 to exon 11 (premature stop 348 codon in exon 12). In addition, the resulting transcript may be a target for non-sense mediated 349 RNA decay and thereby may not be expressed at significant level. In support of this, we were 350 unable to detect faster migrating polypeptides using the antibody raised against residues 44–64 351 (within exon 1-3) of the mouse 3 (Supplementary Fig. 1B). 3 homozygous KO (3-/-) mice were 352 born at expected Mendelian frequency (data not shown). Food intake and spontaneous physical 353 activity, as well as oxygen consumption were similar between wild-type (WT) and 3 KO mice 354 (Supplementary Fig. 1C-E).

355 We first confirmed a complete loss of 3 protein and its associated AMPK catalytic activity in 356 tissues harvested from 3 KO mice (Fig. 1A, B). Expression of 3 is restricted to skeletal muscles

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357 containing a high proportion of glycolytic/fast-twitch fibers [22; 25]. In line with this, we observed 358 that 3 and its associated AMPK activity were predominantly detected in glycolytic gastrocnemius 359 (GAS) and extensor digitorum longus (EDL) muscles in WT mice. A modest expression of 3 and 360 its associated AMPK activity were detected in soleus muscle (which contains a high proportion of 361 oxidative/slow-twitch fibers) from WT mice when 3 proteins were enriched by immunoprecipitation 362 prior to the immunoblotting (Fig. 1A, B). We detected a faint band immuno-reactive to the 3 363 antibody in liver lysates from both WT and 3 KO mice (Fig. 1A, middle panel). We confirmed that 364 the observed band was non-specific as it was readily detected in IgG control samples (Fig. 1A, 365 lower panel) and only a negligible background 3-associated AMPK activity was detected in liver 366 (and also heart) lysates in both WT and 3 KO mice (Fig. 1B). Next, we assessed if loss of 3 367 affected AMPK1- and 2-containing complex activity in GAS muscle. As illustrated in Fig 1C and 368 D, we observed that while AMPK1 activity was unaltered, AMPK2 activity was reduced (50%). 369 Since we and others have shown that 3 predominantly interacts with 2 and 2 [21; 22] to form a 370 stable trimeric α2β2γ3 complex, we hypothesized that a constitutive loss of 3 would cause 371 reduced expressions of 2 and 2 due to their destabilization as monomers. To test this 372 hypothesis, we performed an analysis of AMPK subunit/isoform abundance in both glycolytic (EDL 373 and GAS) and oxidative (soleus) muscles in WT and 3 KO mice (Fig. 1E-G). We previously 374 performed an extensive antibody validation for all AMPK isoforms using individual isoform- 375 specific KO mouse tissues as negative controls and also reported that 2 proteins (UniProt ID: 376 Q91WG5 isoform A) were not detectable in mouse skeletal muscles [22]. Immunoblot analysis 377 revealed that protein levels of 2, total AMPK using a pan 1/2 antibody, and 2 isoforms were 378 selectively reduced (20-30%) in EDL and GAS (Fig. 1E, F), but not in soleus (Fig. 1G), of 3 KO 379 as compared to WT mice. There was no compensatory increase in 1 isoform in 3 KO muscles. 380 To examine if the reduced protein abundance of 2 and 2 was due to decreased mRNA 381 expression of the Prkaa2 and Prkab2 (the genes encoding AMPK2 and 2, respectively) in the 3 382 KO mice, we performed qPCR analyses (Fig. 1H, I). We confirmed that Prkag3 mRNA expression 383 was undetectable in skeletal muscle from 3 KO mice, and observed that there were no differences 384 in mRNA expressions of other AMPK subunit/isoforms in GAS (Fig. 1H) or soleus (Fig. 1I) 385 between WT and 3 KO mice. Taken together, we have demonstrated that a genetic/constitutive 386 loss of AMPK3 causes reductions of AMPK2 and 2 proteins without affecting their mRNA 387 expressions in glycolytic skeletal muscle. 388 389 3.2. AMPK3 deficiency has no impact on mitochondrial content and components or fiber- 390 type composition in skeletal muscle.

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391 A loss-of-function of skeletal muscle AMPK is associated with reduced mitochondrial content and 392 function [45-48]. Interestingly, a transgenic mouse model overexpressing 3 mutant (R225Q, a 393 gain-of-function mutation), was associated with higher mitochondrial content and increased 394 amount of a marker of the oxidative capacity (succinate dehydrogenase) in individual muscle fibers 395 of the white portion of GAS [49]. Nevertheless, 3 deficiency did not cause alterations in 396 mitochondrial content or other parameters in GAS muscle [49]. However, the previously generated 397 3 deficient mice did not exhibit significantly reduced expression or activity of AMPK2 [25], the 398 predominant -catalytic isoform in skeletal muscle. In the current study we wanted to address 399 whether 3 deficiency, coupled to a partial loss of AMPK2 activity (Fig. 1D), had an impact on 400 mitochondrial parameters in both glycolytic (EDL) and oxidative (soleus) skeletal muscle. We 401 observed no differences in mitochondrial DNA copy number (Fig. 2 A, B), citrate synthase activity 402 (Fig. 2 C, D), or components of the mitochondrial respiratory chain complex (Fig. 2E, F) in soleus 403 or EDL muscles of WT and 3 mice. Fiber-type analysis of hindlimb cross sections using 404 immunofluorescence revealed no differences in myosin heavy chain isoform composition in EDL or 405 soleus muscles between the genotypes (Fig. 2G, H). We also observed no difference in skeletal 406 muscle fiber size (cross sectional area) between the genotypes (Fig. 2I). Collectively, we show that 407 constitutive 3 deficiency does not affect mitochondrial content, respiratory chain complex 408 expression, or fiber type composition in both glycolytic and oxidative skeletal muscle. 409 410 3.3. AMPK3 KO mice exhibit normal glucose homeostasis on chow and in response to high 411 fat diet (HFD) feeding. 412 Transgenic mice overexpressing the 3 mutant (R225Q) exhibit an increase in muscle lipid 413 oxidation and are protected against HFD-induced insulin resistance in skeletal muscle [25]. In the 414 current study, we examined if 3 deficiency affected glucose homeostasis under standard chow 415 and in response to HFD feeding. Body weight and composition were similar between WT and 3 416 KO mice during both chow and HFD feeding periods (Fig. 3A, B). We observed similar levels of 417 plasma insulin and leptin on chow diet between WT and 3 KO mice, with their levels increased in 418 a comparable manner for both genotypes in response to HFD feeding (Fig. 3C, D). Consistent with 419 these results, we observed comparable fasted blood glucose levels and no difference in glucose 420 tolerance between the genotypes irrespective of the diets (Fig. 3E). To complement these in vivo 421 results, we assessed insulin signaling and glucose uptake in isolated EDL muscle ex vivo. As 422 shown in Fig. 3F, basal glucose uptake and Akt phosphorylation were comparable between WT 423 and 3 KO mice and insulin equally stimulated both parameters in both genotypes. We also 424 confirmed that there was no difference in the expression of GLUT4 and hexokinase II in EDL

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425 muscle between WT and 3 KO mice (Fig. 3F). Taken together, these results suggest that 3 is 426 dispensable for maintenance of glucose homeostasis on chow and in response to HFD. 427 428 3.4. AMPK3 deficiency causes attenuated AICAR-stimulated glucose uptake in glycolytic 429 skeletal muscle ex vivo and blood glucose lowering in vivo. 430 AICAR-stimulated glucose uptake in skeletal muscle requires functional AMPK [34]. Consistent 431 with previous studies [30; 39], AICAR promoted glucose uptake robustly in EDL (2.5-fold) and 432 modestly in soleus (1.6-fold) ex vivo in WT mice (Fig. 4A, B). Interestingly, we observed that 433 AICAR-stimulated glucose uptake was profoundly reduced in EDL, but not in soleus, in 3 KO mice 434 (Fig. 4A, B). To examine if a loss of 3 affected AICAR-induced AMPK activity, we measured 435 phosphorylation of ACC and TBC1D1, established surrogate markers of cellular AMPK activity in 436 muscle. As shown in Fig. 4C-F, phosphorylation of ACC and TBC1D1 was increased in both EDL 437 and soleus in response to AICAR in WT mice. The AICAR-mediated increase in phosphorylation of 438 ACC and TBC1D1 was reduced in EDL, but not in soleus, in 3 KO mice (Fig. 4C-F). We 439 confirmed that there is no sex-dependent AICAR effect, as AICAR-stimulated glucose uptake was 440 similarly reduced in EDL muscle from female 3 KO mice (data not shown). We wanted to explore 441 if a partial loss of 3 results in a reduction of AICAR-stimulated glucose uptake in EDL. 442 Heterozygous 3+/- mice had ~50% reduction in 3 expression in GAS, but the expression of total 443 AMPK, 2 and 1/2 was not reduced (Supplementary Fig. 2A, B). Incubation of EDL with 444 AICAR ex vivo resulted in similar increases in 3-associated activity and glucose uptake, as well as 445 phosphorylation of ACC and TBC1D1 in both WT and heterozygous 3+/- mice (Supplementary 446 Fig. 2C-G). 447 We next wanted to determine if 3 deficiency affected the hypoglycemic effects of AICAR in vivo. 448 We utilized partially fasted animals (3-hour fast, 07:00-10:00), as the AICAR-induced reduction of 449 blood glucose in overnight fasted (16 hours) mice was predominantly caused by the suppression of 450 hepatic glucose output [13]. After administration of a bolus of AICAR (250 mg/kg body weight, i.p.) 451 or vehicle, we monitored blood glucose kinetics for 2 hours in WT and 3 KO mice. As shown in 452 Fig. 4G, we observed that the blood glucose-lowering action of AICAR was blunted (40 and 60 min 453 time points) in 3 KO compared to WT mice. We confirmed that ZMP content in GAS muscle 454 following AICAR administration was comparably increased between the two genotypes (Fig. 4H). 455 Additionally, AICAR did not affect adenylate energy charge in GAS from both genotypes 456 (Supplementary Fig. 2H). Collectively, these results suggest that 3 (i.e. 3-containing AMPK 457 complex(es)) plays an important role in AICAR-mediated glucose uptake and disposal in glycolytic 458 skeletal muscles.

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459 3.5. ADaM site-targeted activators normally stimulate glucose uptake in skeletal muscle and 460 lower blood glucose levels in AMPK3 KO mice. 461 The ADaM site-binding pan AMPK activator, 991, robustly stimulates glucose uptake in isolated 462 mouse skeletal muscle tissues ex vivo [22; 50]. We initially confirmed that the 991-stimulated 463 glucose uptake was fully dependent on AMPK in both EDL and soleus using skeletal muscle- 464 specific AMPK1/2 double KO (m-1/2 DKO) mice (Fig. 5A, B). Immunoblot analysis validated 465 AMPK deficiency and profound decreases in phosphorylation of ACC and TBC1D1 in the 466 absence or presence of 991 in both EDL and soleus in the m-1/2 DKO mice (Fig. 5C, D). We 467 next assessed the effect of 991, MK-8722 (a structural analogue of 991 [24]), and AICAR on 3- 468 associated AMPK activity in isolated muscle from WT and 3 KO ex vivo. As shown in Fig 5E, 3- 469 associated activity was increased (~1.5-2-fold) with 991 or MK-8722 and robustly increased (~3- 470 fold) with AICAR in EDL from WT mice. Despite minimal 3-associated activity detectable in 471 soleus, the activity was increased 2-fold with 991 in WT mice (Fig. 5F). As expected, there was 472 no 3-associated AMPK activity present in skeletal muscle from 3 KO mice (Fig. 5E, F). In 473 contrast to the effect of AICAR, incubation of EDL with 991 or MK-8722 resulted in comparable 474 increases in glucose uptake in WT and 3 KO mice (Fig. 5G). We also observed that 991- 475 stimulated glucose uptake was similar in soleus between WT and 3 KO mice (Fig. 5H). 991 476 and/or MK-8722 increased phosphorylation of ACC and TBC1D1 in EDL and soleus with no 477 differences in the levels of phosphorylation between WT and 3 KO mice (Fig. 5I-L). Consistent 478 with the ex vivo results, oral administration of MK-8722 (10 or 30 mg/kg body weight) resulted in a 479 comparable blood glucose-lowering kinetics in vivo between WT and 3 KO mice (Fig. 5M and 480 Supplementary Fig. 3). Taken together, we demonstrate that 3 is dispensable for the stimulation 481 of glucose uptake and disposal in skeletal muscle in response to 991 or MK-8722 (ADaM site 482 targeted compounds). 483 484 3.6. AMPK3 protein and its associated AMPK trimeric complexes are present in mouse 485 BAT. 486 Prakg3 mRNA are expressed in mouse brown adipose precursors [37], however whether 3 487 proteins are expressed and exist as part of functional AMPK trimeric complexes in BAT is 488 unknown. We initially performed a comparison of AMPK subunit/isoform protein expression profiles 489 between skeletal muscle (EDL) and BAT from WT mice, which revealed distinct profiles between 490 the two tissues (Fig. 6A). Compared to skeletal muscle, BAT expresses relatively higher and lower 491 amounts of 1 and 2, respectively. The total AMPK content (assessed by a pan-AMPK 492 antibody) was similar between the tissues. However, the efficacy of the isoform-specific detection

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493 of 1 and 2 proteins by this antibody is unknown. We observed a divergent expression pattern of 494 the  isoforms between the tissues. While skeletal muscle predominantly expresses 2, BAT 495 predominantly expresses 1 (Fig. 6A). In contrast, 1 expression is similar between the tissues. 496 We next immunoprecipitated 3 from BAT (and GAS muscle as control) and performed either 3- 497 associated AMPK kinase activity assay or immunoblot analysis to identify  and  subunit isoforms 498 interacting with 3. As shown in Fig. 6B, we detected 3 and its associated AMPK activity in BAT 499 from WT, but not from 3 KO mice. Interestingly, 3 preferentially interacts with 2 and 2 (Fig. 500 6C), whereas 1 interacts with 1/2 and preferentially with 1 (Fig. 6D). We also observed that 3 501 deficiency in BAT did not affect abundance of other AMPK subunit isoforms (Fig. 6E). Collectively, 502 we provide evidence that AMPK3 protein is expressed in BAT and it forms functional complexes 503 by mainly interacting with 2 and 2. 504 505 3.7. AMPK3 is not required for the acute induction of UCP1-mediated non-shivering 506 thermogenesis in the BAT. 507 AMPK plays an important role for BAT formation [37] and thermogenesis in response to cold 508 exposure and 3-adrenoreceptor (3-AR) stimulation in rodents [36]. We probed BAT function 509 using the 3-AR agonist CL-316,243 (CL), which increases thermogenesis through a UCP1- 510 dependent mechanism [44]. A single injection of CL increased oxygen consumption and 511 interscapular BAT surface area temperature in both WT and 3 KO mice, but there were no 512 differences between the genotypes (Fig. 7A-C). Furthermore, CL increased serum non-esterified 513 free fatty acid concentration to a similar extent in both WT and 3 KO mice, indicating no major 514 alterations in lipolysis (Fig. 7D). 515 516 3.8. AMPK3 is not required for the adaptive response to non-shivering thermogenesis or 517 the browning of inguinal white adipose tissue (iWAT). 518 We next performed injections of CL for 5 consecutive days to determine if γ3 is required for the 519 adaptive response to non-shivering thermogenesis or the browning of iWAT. Daily treatment of 520 mice with CL increased oxygen consumption without altering body or BAT weight, but did reduce 521 iWAT weight similarly in both WT and 3 KO mice (Fig. 7E-H). Furthermore, 3 KO mice treated 522 with CL for 5 days had similar morphological changes in BAT – with smaller lipid droplets – and the 523 appearance of multilocular adipocytes within iWAT (Fig. 7I, J). Lastly, CL treatment increased 524 UCP1 expression (at both transcript and protein levels), as well as levels of other thermogenic and 525 mitochondrial genes such as Cox2, Cox8b and Cidea in both WT and 3 KO mice (Fig. 7K-M).

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526 These results demonstrate that γ3 is dispensable for -adrenergic-induced remodeling of BAT and 527 iWAT in mice.

528

529 4. DISCUSSION 530 AMPK has been considered a promising target for the treatment of the metabolic syndrome over 531 the last decades. The identification of new mechanisms for drug-targeting on AMPK (i.e. discovery 532 of ADaM site) has advanced the development of more potent and selective AMPK activators with 533 improved bioavailability [9]. Recent proof of concept studies in rodents and non-human primates 534 have compellingly demonstrated that oral administration of pan AMPK activators (e.g. MK-8722, 535 PF-739) targeting the ADaM site can promote glucose uptake in skeletal muscle, and ameliorate 536 insulin resistance and reduce hyperglycemia without causing hypoglycemia [23; 24]. Since 3 is 537 exclusively expressed in skeletal muscle, understanding of the physiological roles that 3 plays in 538 regulating glucose metabolism/homeostasis is important for the development of skeletal muscle- 539 selective AMPK activators. In addition, a recent in vitro study reports that 3 plays a role in BAT 540 development [37] prompted us to investigate the role for 3 in thermogenesis and adipose 541 browning in vivo. In the current study we found that genetic ablation of 3 resulted in a selective 542 loss of AICAR-, but not MK-8722-induced blood glucose-lowering in vivo and glucose uptake 543 specifically in glycolytic muscles ex vivo. We also found that 3 is dispensable for the acute 544 induction of UCP1-mediated non-shivering thermogenesis in BAT or the adaptive response to non- 545 shivering thermogenesis and the browning of WAT. 546 We observed that the levels of 2 and 2 isoforms were reduced (20-30%) in glycolytic 547 (GAS and EDL), but not in oxidative (soleus) muscle in 3-/- KO compared to WT mice. In soleus, a 548 previous study reported that 3 was only detectable in a complex with 2 and 2, but relative 549 amount of this complex was shown to be only 2% and >90% of 2 and 2 forms complexes with 1 550 [21; 34]. In contrast, 2 and 2 form complexes with 1 (70%) and 3 (20%), respectively in EDL 551 muscle. Therefore, it is plausible that a constitutive deficiency of 3 resulted in a partial loss of 2 552 and 2 proteins due to degradation of excess monomeric forms of 2 and 2 in GAS/EDL, but not 553 in soleus muscle. Consistent with this notion, a constitutive deletion of 1, a ubiquitously expressed 554  isoform across tissues, resulted in much more profound reductions of all its interacting AMPK 555 isoforms (1, 2, 1 and 2) in mouse tissues including skeletal muscle [51]. Even though 556 previous work reported that 3 deficiency did not affect the levels of other AMPK isoforms in GAS 557 muscles, there was an 25% reduction of 2 protein expression in GAS muscles from 3 KO 558 compared to WT mice [25]. It might be the case that it did not reach statistical significance due to

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559 insufficient power. In line with this assumption, phosphorylation of AMPK (Thr172) was reduced 560 in EDL from the same 3 KO mouse model (compared to WT) [52]. 561 A complete loss of functional 2 or 2 was associated with ablated glucose uptake with 562 AICAR in mouse skeletal muscle ex vivo [28; 30; 31]. To our knowledge, whether a partial loss of 563 2 and/or 2 (e.g. in heterozygous 2+/- or 2+/- mice) reduces glucose uptake in EDL with AICAR 564 ex vivo is unknown. However, we have previously demonstrated that a profound reduction (>60%) 565 of 2 activity observed in EDL of the LKB1 hypomorphic mice resulted in comparable AICAR- 566 stimulated glucose uptake and ACC phosphorylation compared to WT mice [4]. Moreover, here we 567 report that 991/MK-8722 stimulates glucose uptake in EDL muscle from the 3 KO mice. 568 Therefore, a partial reduction of 2/2 expression (20-30%) is unlikely to be responsible for the 569 decrease in AICAR-stimulated glucose uptake in 3-deficient EDL muscle. 570 One of the major findings of this study was that 3-deficiency caused blunted glucose 571 uptake in EDL ex vivo and hypoglycemic response in vivo with AICAR, but not with ADaM site- 572 targeted compounds (i.e. 991, MK-8722). This was particularly intriguing as both AICAR and 573 991/MK-8722 require intact AMPK catalytic activity to promote glucose uptake in skeletal muscle 574 tissues/cells [23; 24; 28-32; 45; 50], and we report that both AICAR and 991/MK-8722 increased 575 3-associated AMPK activity. The dose of AICAR (2mM) utilized had a more potent effects on 3- 576 associated activity (~3-fold increase) as compared to 10 M 991/MK-8722 (~1.5-2-fold). However, 577 the results from this assay do not reflect cellular activity, as the in vitro kinase assay following 578 immunoprecipitation accounts for covalently-regulated activity (e.g. phosphorylation), but not 579 allosterically-regulated (i.e. by AMP/ZMP, 991/MK-8722) activity. Judging from phosphorylation 580 levels of ACC and TBC1D1, AICAR and 991/MK-8722 comparably increased cellular AMPK 581 activity in skeletal muscle. However notably, compound-induced phosphorylation of ACC and 582 TBC1D1 ex vivo was only reduced in EDL when treated with AICAR, but not with 991/MK-8722, in 583 3 KO compared to WT. This raises the possibility that AICAR preferentially activates 3- over 1- 584 containing complex(es). Concordantly, AMP appears to have stronger binding affinity to nucleotide 585 binding site 3 (in the CBS domain) of 3 (∼40 μM) than 1 (∼300-600 μM) in vitro (using bacterial 586 AMPK-complex preparations) [53]. On the other hand, another in vitro study reported that while 587 AMP potently (allosterically) activated 221 (~3-fold), it barely activated 223 (<15%) complex 588 [54]. Thus, how these in vitro results can be interpreted and translated into cellular context is 589 unclear. The -isoforms all contain a highly conserved C-terminal region harboring the four CBS 590 domains. Conversely, the γ2 and γ3 isoforms contain long N-terminal extensions that are not 591 present in the γ1 isoform. These N-terminal extensions display no apparent sequence 592 conservation between isoforms. To date, there are no crystal structures available for γ2- or γ3-

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593 containing AMPK trimeric complexes and it is unknown whether the N-terminal extensions of γ2 or 594 γ3 play any functional role. A recent study using cell-based assays demonstrated that 221 and 595 223 complexes were similarly activated in response to 991 treatment, whereas 222 596 complexes exhibited a greater activation (compared to 221/223 complexes) [55]. The 597 authors proposed that the effect is mediated by the N-terminal region of γ2 and is due to enhanced 598 protection of AMPK Thr172 from dephosphorylation. Whether N-terminal extension of 3 has any 599 specific role to play in muscle cells/tissue and in AMP/ZMP-mediated regulation of AMPK and 600 glucose uptake in skeletal muscle is unknown.

601 Even though 3 deficiency was associated with reduced AICAR-stimulated glucose uptake 602 in EDL muscle ex vivo, we provide the first evidence that the AICAR-induced blood glucose 603 lowering effect in vivo was robustly reduced in 3 KO compared to WT mice, which was quite 604 similar to AMPK2 KO and 2 kinase-dead (KD) expressing transgenic mice [28; 30]. Indeed, 2 605 KO and KD mice still showed decreases in blood glucose in response to an acute injection of 606 AICAR, which is most likely due to the inhibitory effect of AICAR on hepatic glucose production 607 through ZMP-dependent inhibition of fructose 1,6-bisphosphatase 1 [13; 56]. MK-8722 has been 608 shown to cause hypoglycemic effect through the stimulation of glucose uptake in both glycolytic 609 (GAS) and oxidative (soleus) muscle in vivo [24]. In contrast to AICAR, but consistent with ex vivo 610 data, we provided evidence that MK-8722-induced skeletal muscle glucose uptake and blood 611 glucose-lowering effects were comparable between 3 KO and WT mice. This compellingly 612 demonstrates that 3 is dispensable (in other words 1 is sufficient) in stimulating glucose uptake in 613 skeletal muscle in response to pan-AMPK ADaM site-binding activators. 614 Evidence suggests that AMPK plays a vital role in regulating the development of BAT, 615 maintenance of BAT mitochondrial function, and browning of WAT [35]. We provided genetic 616 evidence that mice lacking functional AMPK specifically in adipocytes, through an inducible 617 deletion of 1 and 2, were intolerant to cold and resistant to -adrenergic stimulation of brown 618 and beige adipose tissues [36]. Similar findings were also observed in AMPK 1/2 KO mice [57]. 619 Detailed protein expression profiles of AMPK subunit isoforms in BAT in comparison to other 620 tissues have not been performed, and to our knowledge presence of 3 protein in BAT has not 621 been demonstrated. RNA sequencing results identified 3 at intermediate amounts (Reads Per 622 Kilobase of transcript per Million mapped reads (RPKM), >40) in mouse brown preadipocytes [37]. 623 Strikingly, RNAi-mediated knockdown of either 1 or 3 (but not 2) in brown adipocyte precursors 624 was sufficient to profoundly reduce (>80%) UCP1 protein expression. Using 3-specific antibodies 625 we developed, we have demonstrated that 3 protein/activity is present and in complex mainly with 626 2 and 2 in mouse BAT. Even though we showed that 3 is dispensable for -adrenergic-induced

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627 thermogenesis and remodeling of BAT and iWAT, future studies are warranted to determine if the 628 specific activation of the 3-containing complexes (when such drugs are available) induces adipose 629 browning and subsequent amelioration of insulin resistance and fatty liver disease. 630 631 5. CONCLUSIONS 632 We demonstrated that a genetic loss of 3 resulted in a selective loss of AICAR-stimulated 633 glucose-lowering in vivo and glucose uptake specifically in glycolytic skeletal muscles ex vivo. We 634 also showed that 3 is dispensable for thermogenesis and the browning of WAT. The potent pan- 635 AMPK activators targeting the ADaM site are effective in reversing hyperglycemia in rodents and 636 non-human primates, and this is due to activation of AMPK in skeletal muscle, not liver [23; 24]. 637 This might make them valuable adjuncts to metformin, which acts primarily on the liver [13; 58; 59]. 638 However, there are remaining important safety issues that need to be carefully considered and 639 examined, such as the potential for AMPK activation to promote cardiac hypertrophy or the survival 640 of cancer cells (e.g. under hypoxic conditions). To avoid these potential liabilities, the development 641 of AMPK activators that can be targeted to specific tissues (for example, liver, muscle and 642 adipose) by taking advantage of isoform-specific selectivity may be beneficial. We and others have 643 shown that selective targeting of specific AMPK isoforms (e.g. 1, 1) by small molecules is 644 possible [9; 60; 61]. When a 3-complex selective activator is available in the future, ascertaining 645 whether it sufficiently promotes skeletal muscle glucose uptake without causing cardiac 646 hypertrophy and glycogen accumulation will be of interest. 647 648 AUTHOR CONTRIBUTIONS 649 Conceptualization: K.S. Experimental design: P.Rh., E.M.D., P.R., D.A., N.B., J.S., M.D.S., A.J.O., 650 M.F.K., G.R.S., K.S. Experimental execution: P.Rh., E.M.D., P.R., D.A., N.B., J.S., M.D.S., A.J.O., 651 J.M.Y., A.M.E., J.L.S.G., Q.O., M.F.K., M.M. Supervision: J.S., N.J., J.S.O., J.T.T., P.M., J.W.S., 652 M.J.S, P.D., S.C., G.R.S., K.S. Writing- Original draft preparation: K.S., P.Rh. Writing- Reviewing 653 and Editing: All authors. 654 655 GRANTS 656 The work is supported by the Novo Nordisk Foundation (NNF20OC0063515) to K.S. 657 E.M.D. is a Vanier Canada Graduate Scholar. GRS is supported by a Diabetes Canada 658 Investigator Award (DI-5-17-5302-GS), a Canadian Institutes of Health Research 659 Foundation Grant (201709FDN-CEBA-116200), a Tier 1 Canada Research Chair and a 660 J. Bruce Duncan Endowed Chair in Metabolic Diseases. The P.M. lab is funded by the 661 Association Française contre les Myopathies (AFM n°21711), and by the Agence

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662 Nationale pour la Recherche (Myolinc, ANR R17062KK). J.W.S. and J.S.O. were 663 supported by National Health and Medical Research Council (NHMRC) project grants 664 (GNT1138102 and GNT1145265, respectively). This project was supported in part by 665 the Victorian Government’s Operational Infrastructure Support Program. A.J.O. is 666 supported by a PhD scholarship funded by the Australian Catholic University. M.F.K. 667 has received funding from Danish Diabetes Academy and Novo Nordisk Foundation. 668 Novo Nordisk Foundation Center for Basic Metabolic Research is an independent 669 Research Center based at the University of Copenhagen, Denmark, and partially 670 funded by an unconditional donation from the Novo Nordisk Foundation (Grant number 671 NNF18CC0034900). 672 673 ACKNOWLEDGMENTS 674 We thank Carles Canto, Magali Joffraud, Guillaume Jacot, Maria Deak, Caterina Collodet, Alix 675 Zollinger, Sylviane Metairon, and Stefan Christen (all affiliated with Nestlé Research) for their 676 technical assistance and input for experimental design, assays, and data analysis/interpretation. 677 We also thank Juleen Zierath for her critical review of the manuscript. 678 679 CONFLICT OF INTEREST 680 P.Rh., P.D., J.S., M.J.S., J.L.S.G., M.M. are current and N.B. and K.S. were former employees of 681 the Nestlé Research (Switzerland). 682

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683 REFERENCES 684 [1] Hardie, D.G., 2011. AMP-activated protein kinase: an energy sensor that regulates all 685 aspects of cell function. Genes Dev 25(18):1895-1908. 686 [2] Herzig, S., Shaw, R.J., 2018. AMPK: guardian of metabolism and mitochondrial homeostasis. 687 Nat Rev Mol Cell Biol 19(2):121-135. 688 [3] Hawley, S.A., Davison, M., Woods, A., Davies, S.P., Beri, R.K., Carling, D., et al., 1996. 689 Characterization of the AMP-activated protein kinase kinase from rat liver and identification of 690 threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase. J Biol 691 Chem 271(44):27879-27887. 692 [4] Sakamoto, K., McCarthy, A., Smith, D., Green, K.A., Grahame Hardie, D., Ashworth, A., et 693 al., 2005. Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake 694 during contraction. EMBO J 24(10):1810-1820. 695 [5] Shaw, R.J., Lamia, K.A., Vasquez, D., Koo, S.H., Bardeesy, N., Depinho, R.A., et al., 2005. The 696 kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 697 310(5754):1642-1646. 698 [6] Gowans, G.J., Hawley, S.A., Ross, F.A., Hardie, D.G., 2013. AMP is a true physiological 699 regulator of AMP-activated protein kinase by both allosteric activation and enhancing net 700 phosphorylation. Cell Metab 18(4):556-566. 701 [7] Xiao, B., Sanders, M.J., Underwood, E., Heath, R., Mayer, F.V., Carmena, D., et al., 2011. 702 Structure of mammalian AMPK and its regulation by ADP. Nature 472(7342):230-233. 703 [8] Oakhill, J.S., Steel, R., Chen, Z.P., Scott, J.W., Ling, N., Tam, S., et al., 2011. AMPK is a direct 704 adenylate charge-regulated protein kinase. Science 332(6036):1433-1435. 705 [9] Steinberg, G.R., Carling, D., 2019. AMP-activated protein kinase: the current landscape for 706 drug development. Nat Rev Drug Discov 18(7):527-551. 707 [10] Buhl, E.S., Jessen, N., Pold, R., Ledet, T., Flyvbjerg, A., Pedersen, S.B., et al., 2002. Long- 708 term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats 709 displaying features of the insulin resistance syndrome. Diabetes 51(7):2199-2206. 710 [11] Guigas, B., Sakamoto, K., Taleux, N., Reyna, S.M., Musi, N., Viollet, B., et al., 2009. Beyond 711 AICA riboside: in search of new specific AMP-activated protein kinase activators. IUBMB Life 712 61(1):18-26. 713 [12] Foretz, M., Hebrard, S., Leclerc, J., Zarrinpashneh, E., Soty, M., Mithieux, G., et al., 2010. 714 Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via 715 a decrease in hepatic energy state. J Clin Invest 120(7):2355-2369. 716 [13] Hunter, R.W., Hughey, C.C., Lantier, L., Sundelin, E.I., Peggie, M., Zeqiraj, E., et al., 2018. 717 Metformin reduces liver glucose production by inhibition of fructose-1-6-bisphosphatase. Nat Med 718 24(9):1395-1406. 719 [14] Collodet, C., Foretz, M., Deak, M., Bultot, L., Metairon, S., Viollet, B., et al., 2019. AMPK 720 promotes induction of the tumor suppressor FLCN through activation of TFEB independently of 721 mTOR. FASEB J 33(11):12374-12391. 722 [15] Cool, B., Zinker, B., Chiou, W., Kifle, L., Cao, N., Perham, M., et al., 2006. Identification and 723 characterization of a small molecule AMPK activator that treats key components of type 2 724 diabetes and the metabolic syndrome. Cell Metab 3(6):403-416. 725 [16] Goransson, O., McBride, A., Hawley, S.A., Ross, F.A., Shpiro, N., Foretz, M., et al., 2007. 726 Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase. J 727 Biol Chem 282(45):32549-32560.

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816 [47] Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J.T., Pehmoller, C., et al., 2014. AMPK 817 controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. 818 FASEB J 28(7):3211-3224. 819 [48] Zong, H., Ren, J.M., Young, L.H., Pypaert, M., Mu, J., Birnbaum, M.J., et al., 2002. AMP 820 kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy 821 deprivation. Proc Natl Acad Sci U S A 99(25):15983-15987. 822 [49] Garcia-Roves, P.M., Osler, M.E., Holmstrom, M.H., Zierath, J.R., 2008. Gain-of-function 823 R225Q mutation in AMP-activated protein kinase gamma3 subunit increases mitochondrial 824 biogenesis in glycolytic skeletal muscle. J Biol Chem 283(51):35724-35734. 825 [50] Lai, Y.C., Kviklyte, S., Vertommen, D., Lantier, L., Foretz, M., Viollet, B., et al., 2014. A small- 826 molecule benzimidazole derivative that potently activates AMPK to increase glucose transport in 827 skeletal muscle: comparison with effects of contraction and other AMPK activators. Biochem J 828 460(3):363-375. 829 [51] Foretz, M., Hebrard, S., Guihard, S., Leclerc, J., Do Cruzeiro, M., Hamard, G., et al., 2011. 830 The AMPKgamma1 subunit plays an essential role in erythrocyte membrane elasticity, and its 831 genetic inactivation induces splenomegaly and anemia. FASEB J 25(1):337-347. 832 [52] Treebak, J.T., Birk, J.B., Rose, A.J., Kiens, B., Richter, E.A., Wojtaszewski, J.F., 2007. AS160 833 phosphorylation is associated with activation of alpha2beta2gamma1- but not 834 alpha2beta2gamma3-AMPK trimeric complex in skeletal muscle during exercise in humans. Am J 835 Physiol Endocrinol Metab 292(3):E715-722. 836 [53] Rajamohan, F., Reyes, A.R., Frisbie, R.K., Hoth, L.R., Sahasrabudhe, P., Magyar, R., et al., 837 2016. Probing the enzyme kinetics, allosteric modulation and activation of alpha1- and alpha2- 838 subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by 839 pharmacological and physiological activators. Biochem J 473(5):581-592. 840 [54] Ross, F.A., Jensen, T.E., Hardie, D.G., 2016. Differential regulation by AMP and ADP of 841 AMPK complexes containing different gamma subunit isoforms. Biochem J 473(2):189-199. 842 [55] Willows, R., Navaratnam, N., Lima, A., Read, J., Carling, D., 2017. Effect of different gamma- 843 subunit isoforms on the regulation of AMPK. Biochem J 474(10):1741-1754. 844 [56] Vincent, M.F., Erion, M.D., Gruber, H.E., Van den Berghe, G., 1996. Hypoglycaemic effect of 845 AICAriboside in mice. Diabetologia 39(10):1148-1155. 846 [57] Wu, L., Zhang, L., Li, B., Jiang, H., Duan, Y., Xie, Z., et al., 2018. AMP-Activated Protein 847 Kinase (AMPK) Regulates Energy Metabolism through Modulating Thermogenesis in Adipose 848 Tissue. Front Physiol 9:122. 849 [58] Fullerton, M.D., Galic, S., Marcinko, K., Sikkema, S., Pulinilkunnil, T., Chen, Z.P., et al., 2013. 850 Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing 851 effects of metformin. Nat Med 19(12):1649-1654. 852 [59] Rena, G., Pearson, E.R., Sakamoto, K., 2013. Molecular mechanism of action of metformin: 853 old or new insights? Diabetologia 56(9):1898-1906. 854 [60] Hunter, R.W., Foretz, M., Bultot, L., Fullerton, M.D., Deak, M., Ross, F.A., et al., 2014. 855 Mechanism of action of compound-13: an alpha1-selective small molecule activator of AMPK. 856 Chem Biol 21(7):866-879. 857 [61] Langendorf, C.G., Ngoei, K.R.W., Scott, J.W., Ling, N.X.Y., Issa, S.M.A., Gorman, M.A., et al., 858 2016. Structural basis of allosteric and synergistic activation of AMPK by furan-2-phosphonic 859 derivative C2 binding. Nat Commun 7:10912. 860 861

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862 FIGURE LEGENDS 863 Figure 1: Genetic ablation of the AMPKγ3 causes a significant loss of α2 and β2 expression 864 in mouse glycolytic skeletal muscles 865 (A) Immunoblot (IB) analysis of γ3 expression in a panel of tissues extracted from wild-type (WT) 866 or AMPKγ3-null (γ3-/-) mice (upper panel). γ3 expression was further analyzed by immunoblotting 867 following enrichment of the γ3 proteins via immunoprecipitation (IP) from the indicated tissue 868 extracts (200 g) (middle panel). Liver and skeletal muscle (GAS) tissue extracts from the 869 indicated genotypes were used for immunoprecipitation with either γ3-specific antibody or species- 870 matched IgG (as negative control) and the immune-complexes were subsequently immunoblotted 871 with γ3 antibody (lower panel). (B) The γ3-containing AMPK complexes were immunoprecipitated 872 from the indicated tissues harvested from the indicated genotypes and an in vitro AMPK activity 873 assay was performed in duplicate (n=3 per tissue/genotype). (C, D) The In vitro AMPK activity 874 assay was performed on α1- or α2-contaning AMPK complexes immunoprecipitated from GAS 875 extracts (n=9-10 per tissue/genotype). (E-G) Representative immunoblot images and quantification 876 of the AMPK isoform-specific expression using an automated capillary immunoblotting system 877 (Sally Sue) with the indicated antibodies as described in Materials and Methods. AMPK isoform 878 expressions were normalized by their respective vinculin expression (loading control) and are 879 shown as fold change relative to WT. Note that AMPKγ1 expression was quantified using another 880 immunoblotting system (Li-COR, described in the Materials and Methods) due to antibody 881 compatibility (n=5-11 per tissue/genotype). (H, I) Relative levels of mRNA of the indicated genes 882 (encoding AMPK isoforms) in the indicated skeletal muscles were assessed by qPCR (n=5 per 883 tissue/genotype). Results are shown as means ± SEM. Statistical significance was determined 884 using the unpaired, two-tailed Student’s t-test and are shown as #P < 0.05 (WT vs. γ3-/-). GAS; 885 gastrocnemius, EDL; extensor digitorum longus, SOL; soleus, IgG; immunoglobulin G. 886 887 Figure 2: AMPKγ3 deficiency does not affect mitochondrial content and components, or 888 fiber-type composition in skeletal muscles 889 (A, B) Relative quantification of mitochondrial DNA (mtDNA) was performed using qPCR-based 890 assay as described in the Materials and Methods (n=5 per tissue/genotype). (C, D) Citrate 891 synthase activity was measured in the indicated muscle extracts (n=8 per tissue/genotype). (E, F) 892 Immunoblot analysis and quantification of mitochondrial complexes in the indicated muscles (n=7 893 per tissue/genotype). (G-I) Representative cross-sectional images (of n=4 per genotype) of the 894 whole-hindlimb muscle fiber-type analysis of the indicated genotypes using isoform-specific myosin 895 heavy chain (MyHC) and laminin antibodies followed by immunofluorescent signal detection (G). 896 Scale bar=1 mm. Quantification of relative isoform-specific myosin heavy chain (MyHC)

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897 composition/fraction (red: MyHC I, green: MyHC IIa, blue: MyHC IIb, laminin: gray/white) and fiber 898 area in the indicated muscles were performed as described in Materials and Methods. Unstained 899 fibers are not included in the fiber fraction analysis (H, L, n=3-4 per tissue/genotype). Results are 900 shown as means ± SEM. GAS; gastrocnemius, EDL; extensor digitorum longus; SOL; soleus, Tib; 901 tibialis anterior, Plant; plantaris, F; fibula, T; tibia. 902 903 Figure 3: AMPKγ3 is dispensable for maintaining glucose homeostasis under chow and 904 high fat diet (HFD) feeding 905 (A) Time sequence of the diet intervention, analysis of body composition (qNMR), oral glucose 906 tolerance test (GTT) and plasma hormone analysis (blood chemistry). Mice were fed chow diet 907 after weaning until 11 weeks of age before switching to HFD (60% kcal% fat). Body weight over 908 time of the indicated genotypes (n=10 per genotype). (B) Body composition determined by qNMR 909 in the indicated genotypes during the indicated diet treatment. (C, D) Plasma insulin and leptin 910 levels were determined using the commercial enzyme-linked immunosorbent assay kits. (E) Mice 911 were fasted overnight and an oral GTT test was performed during chow (week 10) and HFD (week 912 17) feeding by monitoring blood glucose kinetics over the indicated duration following an oral 913 administration of a bolus of glucose solution (2 g/kg body weight). (F) Extensor digitorum longus 914 (EDL) muscles from the indicated genotypes on chow diet (10-12-week old males from a separate 915 cohort. n=5-7 per genotype) were isolated in incubated in the presence or absence of insulin (100 916 nM) for 50 min and were subjected to glucose uptake assay and immunoblot analysis using the 917 indicated antibodies. Results are shown as means ± SEM. Statistical significance was determined 918 using the unpaired/two-tailed Student’s t-test or one-way analysis of variance with Bonferroni 919 correction and are shown as *P < 0.05 (treatment effect within the same genotype). 920 921 Figure 4: AMPKγ3 is required for AICAR-induced glucose uptake in glycolytic skeletal 922 muscles and hypoglycemia 923 (A-F) EDL or SOL muscles were isolated from the indicated genotypes and incubated in the 924 absence (vehicle, DMSO) or presence of AICAR (2 mM) for 50 min followed by an additional 10- 925 min incubation with the radioactive 2-deoxy-glucose tracer. One portion of the muscle extracts was 926 subjected to glucose uptake measurement (A, B) and the other was used for immunoblot analysis 927 using the automated capillary immunoblotting system with the indicated antibodies (C-F) (n=4-7 928 per treatment/genotype). (G, H) AICAR tolerance test and muscle ZMP analysis. Mice were fasted 929 for 3 hours and injected either with vehicle (water) or AICAR (250 mg/kg body weight, i.p.) followed 930 by blood glucose kinetics measurement over the indicated duration (G). Following the AICAR 931 tolerance test, mice were euthanized and GAS muscles were extracted and ZMP levels were

27 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

932 determined (H) (n=5-12 per treatment/genotype). Results are shown as means ± SEM. Statistical 933 significance was determined using the unpaired/two-tailed Student’s t-test or one-way analysis of 934 variance with Bonferroni correction and are shown as *P < 0.05 (treatment effect within the same 935 genotype), #P < 0.05 (WT vs. γ3-/- within the same treatment). GAS; gastrocnemius, EDL; extensor 936 digitorum longus, SOL; soleus, AICAR; 5-aminoimidazole-4-carboxamide ribonucleoside, ZMP; 937 AICAR monophosphate 938 939 Figure 5: AMPK1/2, but not γ3, is required for glucose uptake skeletal muscles and 940 hypoglycemia in response to the ADaM site-targeted activators, 991 and MK-8722 941 (A-D) EDL or SOL muscles were isolated from the indicated genotypes and incubated in the 942 absence (vehicle, DMSO) or presence of 991 (10 M) for 50 min followed by an additional 10-min 943 incubation with the radioactive 2-deoxy-glucose tracer. One portion of the muscle extracts was 944 subjected to glucose uptake measurement (A, B) and the other was used for immunoblot analysis 945 using the indicated antibodies (followed by a signal detection using enhanced chemiluminescence) 946 (C, D, n=3-4 per treatment/genotype). (E-L) EDL or SOL muscles were isolated from the indicated 947 genotypes and incubated in the absence (vehicle, DMSO) or presence of the indicated compounds 948 for 50 min followed by an additional 10-min incubation with the radioactive 2-deoxy-glucose tracer. 949 One portion of the muscle extracts was subjected to immunoprecipitation with the γ3 antibody 950 followed by an in vitro AMPK activity assay (E, F, n=4-14). The other portion was subjected to 951 glucose uptake measurement (G, H, n=4-9) or immunoblot analysis using the automated capillary 952 immunoblotting system with the indicated antibodies (I-L, n=4-9). M) MK-8722 tolerance test. Mice 953 were fasted for 3 hours and orally treated either with vehicle or MK-8722 (10 mg/kg body weight) 954 followed by blood glucose kinetics monitoring over the indicated duration. Results are shown as 955 means ± SEM. Statistical significance was determined using the unpaired/two-tailed Student’s t- 956 test or one-way analysis of variance with Bonferroni correction and are shown as *P < 0.05 957 (treatment effect within the same genotype), #P < 0.05 (WT vs. γ3-/- within the same treatment). 958 EDL; extensor digitorum longus; SOL; soleus, AICAR; 5-aminoimidazole-4-carboxamide 959 ribonucleoside 960 961 Figure 6: AMPKγ3 is expressed and forms functional trimeric complexes in mouse brown 962 adipose tissue (BAT) 963 (A) Immunoblot analysis of the skeletal muscle (EDL) and BAT extracts harvested from WT mice 964 using the automated capillary immunoblotting system with the indicated antibodies. Note that γ1 965 expression was quantified using another immunoblotting system (Li-COR) due to antibody 966 compatibility. (B) Extracts from GAS muscle (100 g) or BAT (1000 g) were subjected to

28 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

967 immunoprecipitation with 3 antibody and the 3-containing immune-complexes were assayed for 968 AMPK activity in vitro. (C, D) 3- or 1-containing AMPK complexes were immunoprecipitated from 969 GAS (100 g) or BAT (1000 g) extracts and subsequently subjected to immunoblot analysis using 970 the indicated antibodies followed by a signal detection using enhanced chemiluminescence. (E) 971 Quantification of the isoform-specific AMPK expression of a panel of tissues (harvested from WT 972 or 3-/- mice) was performed using the automated capillary immunoblotting system with the 973 indicated antibodies. Results are shown as means ± SEM (n=5-7). GAS; gastrocnemius, EDL; 974 extensor digitorum longus 975 976 Figure 7: AMPK γ3 is not required for the non-shivering thermogenesis or the browning of 977 inguinal white adipose tissue (WAT) in mice

978 (A) Oxygen consumption (VO2), (B, C) Interscapular brown adipose tissue (BAT) surface area 979 temperature with representative thermal images, and (D) serum non-esterified free fatty acid 980 (NEFA) concentration in response to a single injection of saline or CL 316,243 in male WT or γ3-/- 981 mice (0.033 nmol/g, 20 min time-point), n=9-13 per group. Data are means ± SEM with a CL 982 316,243 effect shown as *P < 0.05, as determined via repeated measures two-way analysis of

983 variance (ANOVA). (E) Oxygen consumption (VO2) basally and 6-h post-injection of saline or CL 984 316,243 in male WT or γ3-/- mice on indicated days, n=5-8 per group. (F) Final body weight (BW), 985 (G) BAT weight, and (H) inguinal WAT (iWAT) depot weight following 5 consecutive days of saline 986 or CL 316,243 (5D CL) injections in male WT or γ3-/- mice, n=5-8 per group. (I, J) Representative 987 histological images of H&E-stained BAT (I) and iWAT (J) (10X magnification) from male WT or γ3-/- 988 mice treated with saline or 5D CL. K) mRNA expression of genes indicative of iWAT browning, MT- 989 CO2 (n=4-8 per group), Cox8b (n=5-8 per group), and Cidea (n=5-7 per group) in male WT or γ3-/- 990 mice treated with saline or 5D CL for 5 days. L) Immunoblot analysis and densitometry 991 quantification (M) of UCP1 in male WT and γ3-/- mice treated with saline or 5D CL for 5 days (n=6- 992 8 per group). Data are means ± SEM with *P < 0.05 denoting a 5D CL effect, as determined via 993 repeated measures two-way ANOVA (A) and regular two-way ANOVA. 994 995

29 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

996 Supplementary Figure 1: Generation and general characterization of the AMPKγ3 knockout 997 (KO) mice 998 (A) A schematic illustrating the targeting strategy used to generate Prkag3 knockout (AMPKγ3 999 KO) mouse model (C57BL/6 background). The targeting strategy is based on NCBI transcript 1000 NM_153744_3. The constitutive KO allele is obtained after in-vivo Cre-mediated recombination 1001 using Cre-Deleter mice (Taconic Biosciences) in which Cre is expressed under the control of the 1002 Gt(ROSA)26Sor gene. Deletion of exons 5-10 should result in the loss of function of the Prkag3 1003 gene by deleting the cystathionine -synthase (CBS) 2 domain and parts of the CBS 1 and 3 1004 domains and by generating a frame shift from exon 4 to exon 11 (premature stop codon in exon 1005 12). In addition, the resulting transcript may be a target for Non-sense Mediated RNA Decay and 1006 may, therefore, not be expressed at significant level. (B) Immunoblot analysis of the gastrocnemius 1007 (GAS) muscle extracts obtained from the indicated genotypes using the anti-γ3 antibody raised 1008 against residues 44–64 (within exon 1-3) of the mouse 3. (n=3 per genotype) (C-E) General 1009 mouse phenotyping was performed by PHENOMIN (Illkirch, France). Mice were housed in 1010 metabolic phenotyping cages (TSE system, Labmaster, Germany) and after a 3-hour 1011 acclimatization period at ambient temperature (21°C ± 2), food intake (C), ambulatory activity (D) 1012 and oxygen consumption (E) were monitored. (n=10 per genotype) Results are shown as 1013 means ± SEM. 1014 1015 Supplementary Figure 2: AMPK isoform expression, γ3 activity, AICAR-stimulated glucose 1016 uptake and AMPK signaling in heterozygous γ3+/- mice and muscle energy charge following 1017 AICAR injection in WT and γ3-/- mice 1018 (A, B) Immunoblot analysis and quantification of the AMPK isoform expression in extensor 1019 digitorum longus (EDL) muscles from wild-type (WT) and heterozygous γ3+/- mice using the 1020 indicated antibodies. Representative blot images shown (n=5-6 per genotype). (C, D) γ3-containing 1021 complexes were immunoprecipitated and were subjected to an in vitro AMPK assay. The activity 1022 was shown as absolute unit (C) or fold increase relative to vehicle for corresponding genotype (D). 1023 (n=5-6 per treatment/genotype) (E-G) EDL muscles were isolated from the indicated genotypes 1024 and incubated in the absence (vehicle, DMSO) or presence of AICAR (2 mM) for 50 min followed 1025 by an additional 10-min incubation with the radioactive 2-deoxy-glucose tracer. One portion of the 1026 muscle extracts was subjected to glucose uptake measurement (E) and the other was used for 1027 immunoblot analysis using the automated capillary immunoblotting system with the indicated 1028 antibodies (F. G) (n=5-7 per treatment/genotype). H) Following the AICAR tolerance test (Fig. 4G), 1029 mice were euthanized and GAS muscles were extracted and nucleotide levels were determined for 1030 calculation of the energy charge. (n=5-12 per treatment/genotype. Results are shown as

30 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1031 means ± SEM. Statistical significance was determined using the unpaired/two-tailed Student’s t- 1032 test or one-way analysis of variance with Bonferroni correction and are shown as *P < 0.05 1033 (treatment effect within the same genotype), #P < 0.05 (WT vs. γ3+/- within the same treatment). 1034 1035 Supplementary Figure 3: MK-8722 tolerance test in WT and γ3+/- mice 1036 Mice were fasted for 3 hours and orally treated with MK-8722 (30 mg/kg body weight) followed by 1037 blood glucose kinetics monitoring over the indicated duration.(n=8-10 per treatment/genotype) 1038 Results are shown as means ± SEM. 1039 1040 1041

31 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 1

GAS EDL SOL Liver Heart AMPKγ3 activity A -/- -/- -/- -/- -/- B 6

WT γ3 WTγ3 WTγ3 WTγ3 WT γ3 -1 75 kDa AMPKγ3 (YZ4698)

∙mg WT

IB -1 GAPDH -/- 37 kDa γ3

∙ min 4 IgG short 75 kDa exposure IP: AMPKγ3 (S673D) long 75 kDa exposure 2

IP: IgG γ3 IgG γ3 IgG γ3 γ3 75 kDa IB: AMPKγ3 (YZ4698) pmol Pi incorporated 0 WT γ3-/- WT γ3-/- GAS EDL SOL Liver Heart Liver GAS

AMPKα1 activity AMPKα2 activity

C -1 D 10 -1 25 ∙mg ∙mg -1 8 -1 20

6 15 *

4 10

2 5 pmol Pi incorporated ∙ min 0 pmol Pi incorporated ∙ min 0 WT γ3-/- WT γ3-/-

Protein expression Protein expression Protein expression E (EDL) F (GAS) G (SOL) WT γ3-/- WT γ3-/- WT γ3-/- AMPKα 66 kDa 66 kDa 66 kDa AMPKα1 66 kDa 66 kDa 66 kDa AMPKα2 66 kDa 66 kDa 66 kDa

β1 AMPK β2 40 kDa 40 kDa 40 kDa 37 kDa 37 kDa 37 kDa AMPKγ1 Vinculin 116 kDa 116 kDa 116 kDa

WT WT WT 1.5 1.5 1.5 γ3-/- γ3-/- γ3-/-

1.0 1.0 1.0 # # # # # # 0.5 0.5 0.5 (AMPK/Vinculin) (AMPK/Vinculin) (AMPK/Vinculin) Fold change relative to WT Fold change relative to WT Fold change relative to WT 0 0 0 α α1 α2 β1 γ1β2 α α1 α2 β1 γ1β2 α α1 α2 β1 γ1β2 AMPK subunits AMPK subunits AMPK subunits

H mRNA expression I mRNA expression 1.5 (GAS) WT 1.5 (SOL) WT γ3-/- γ3-/-

1.0 1.0

0.5 0.5 Relative level mRNA Relative level mRNA

0.0 0.0 Prkaa1 Prkaa2 Prkab1 Prkab2 Prkag1 Prkag3 Prkaa1 Prkaa2 Prkab1 Prkab2 Prkag1 Prkag3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 2

Mitochondrial DNA Mitochondrial DNA A 1.5 (EDL) B 1.5 (SOL)

1.0 1.0

0.5 0.5

Relative mtDNA/nDNA ratio Relative mtDNA/nDNA 0 ratio Relative mtDNA/nDNA 0 WT γ3-/- WT γ3-/-

Citrate synthase activity Citrate synthase activity C 400 (EDL) D 500 (SOL) -1

-1 400 300 ∙ mg ∙ mg -1 300 -1 200 200 100

μmol ∙ min 100 μmol ∙ min

0 0 WT γ3-/- WT γ3-/- -/- WT E EDL WT EDL γ3 2 EDL γ3-/- Complex V (ATP5A) 50 kDa 37 kDa Complex IV (MTCO1)

50 kDa Complex III (UQRC2) 1

Complex II (SDH8) 25 kDa 20 kDa Protein levels Complex I (NDUFB8)

150 kDa (Fold Change relative to WT) Vinculin 0 Complex I II III IV V

-/- F SOL WT SOL γ3 2 SOL Complex V (ATP5A) WT 50 kDa -/- 37 kDa γ3 Complex IV (MTCO1)

Complex III (UQRC2) 50 kDa 1

Complex II (SDH8) 25 kDa

20 kDa Protein levels Complex I (NDUFB8)

150 kDa (Fold Change relative to WT) Vinculin 0 Complex I II III IV V

G WT γ3-/- H Fiber fraction Fiber fraction 0.6 (EDL) 0.8 (SOL) WT WT γ3-/- γ3-/- 0.6 0.4

0.4

0.2

Fiber fraction 0.2 Fiber fraction

0 0 MyHC I MyHC IIa MyHC IIb MyHC I MyHC IIa MyHC IIb I Fiber area Fiber area 1500 1500 (EDL) (SOL)

1000 1000 2 2 μ m μ m 500 500

0 0 WT γ3-/- WT γ3-/- bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 3

Blood chemistry qNMR, Blood A chemistry B GTT GTT qNMR Body composition

7 8 9 10 11 12 13 14 15 16 17 18 19 50 25 Chow HFD WT γ3-/- 40 20

15 30 10 Mass (g) 20 Body mass (g) WT 5 γ3-/- 10 0 Chow HFD Chow HFD Chow HFD 0 7 8 9 10 11 12 13 14 15 16 17 18 19 Lean mass Fat mass Body fluids

Age (weeks)

C Plasma insulin D Plasma leptin 6 8 * * * 6 4 -1 -1 * 4 μ g · L

2 ng · mL 2

0 0 WT γ3-/- WT γ3-/- WT γ3-/- WT γ3-/-

Chow HFD Chow HFD

Basal Insulin E Glucose tolerance test F 2.0 WT HFD 25 ) -/- -1 * γ3 HFD 1.5 * · h WT Chow -1 20 1.0 γ 3-/- Chow

15 (μMol · g 0.5 Glucose uptake

10 0 WT γ3-/- WT γ3-/- 66 kDa pAkt (Ser473)

Blood glucose (mM) 5 66 kDa Akt 0 116 kDa 0 15 30 60 120 HXK II GLUT4 Time (min) 40 kDa GAPDH 40 kDa bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 4

A Ex vivo EDL B Ex vivo SOL 2.5 2.0 *

) 2.0 ) * -1 -1 1.5 *

· h *# · h -1 1.5 *# -1 1.0 1.0 (μMol · g (μMol · g

Glucose uptake Glucose uptake 0.5 0.5

0 0 WT γ3-/- WT γ3-/- WT γ3-/- WT γ3-/- Vehicle AICAR Vehicle AICAR

pS212-ACC pS212-ACC C 1.5 (EDL) D 1.5 (SOL) * *# * 1.0 1.0 *

0.5 0.5 S212- ACC/ACC S212-ACC/ACC p p

0 0 WT γ3-/- WT γ3-/- WT γ3-/- WT γ3-/- Vehicle AICAR Vehicle AICAR

pS231-TBC1D1 pS231-TBC1D1 (EDL) (SOL) E 1.5 F 1.5 * * *

1.0 *# 1.0

0.5 0.5 S231-TBC1D1/TBC1D1 S231- TBC1D1/TBC1D1 p p 0 0 -/- -/- WT γ3 WT γ3 WT γ3-/- WT γ3-/- Vehicle AICAR Vehicle AICAR

G AICAR tolerance test H 15 Muscle ZMP content 400 * *

10 # 300 protein -1 # 200 WT Vehicle 5 γ3-/- 100 Blood glucose (mM) WT pmol · mg AICAR γ3-/- 0 0 0 50 100 150 WT γ3-/- WT γ3-/- Time (min) Vehicle AICAR bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Figure 5

Ex vivo EDL Ex vivo SOL A 1.5 B 1.0 * )

) * 0.8 -1 -1 # 1.0 · h · h 0.6 -1 -1 # 0.4 0.5 (μMol · g

(μMol · g 0.2 Glucose uptake Glucose uptake

0 0 m-α1α2+/+ m-α1α2-/- m-α1α2+/+ m-α1α2-/- m-α1α2+/+ m-α1α2-/- m-α1α2+/+ m-α1α2-/- Vehicle 991 Vehicle 991

C m-α1α2+/+ m-α1α2-/- D m-α1α2+/+ m-α1α2-/-

Vehicle 991 Vehicle 991 Vehicle 991 Vehicle 991 75 kDa 75 kDa pAMPK (T172) pAMPK (T172) 75 kDa 75 kDa AMPKα AMPKα pTBC1D1 (S231) 150 kDa pTBC1D1 (S231) 150 kDa TBC1D1 150 kDa TBC1D1 150 kDa pACC (S212) 250 kDa pACC (S212) 250 kDa ACC 250 kDa ACC 250 kDa

GAPDH 37 kDa GAPDH 37 kDa

E EDL F ) SOL ) -1

-1 60 3 ∙mg ∙mg -1

-1 * 40 2 * *

20 * 1 AMPK γ 3 activity AMPK γ 3 activity (pmol Pi incorporated ∙ min

(pmol Pi incorporated ∙ min 0 0 WTγ3-/- WT γ3-/- WTγ3-/- WT γ3-/- WT γ3-/- WT γ3-/- Vehicle 991 MK-8722 AICAR Vehicle 991 (10 μM) (10 μM) (2 mM) G Ex vivo EDL H Ex vivo SOL 2.5 2.5 * * * ** 2.0 * 2.0 ) ) -1 -1 · h

1.5 · h 1.5 -1 -1

1.0 1.0 (μMol · g (μMol · g Glucose uptake 0.5 Glucose uptake 0.5

0 0 WT γ3-/- WT γ3-/- WT γ3-/- WT γ3-/- WT γ3-/-

Vehicle 991 MK-8722 Vehicle 991 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

I pS212-ACC J pS212-ACC (EDL) (SOL) 1.5 1.0 * * * * * 0.8 1.0 * 0.6

0.4 0.5 S212-ACC/ACC S212-ACC/ACC p p 0.2

0 0 -/- -/- -/- WTγ3 WT γ3 WT γ3 WT γ3-/- WT γ3-/- Vehicle 991 MK-8722 Vehicle 991

K pS231-TBC1D1 L pS231-TBC1D1 1.5 (EDL) 1.5 (SOL)

* * * * * 1.0 * 1.0

0.5 0.5 S231- TBC1D1/TBC1D1 p S231- TBC1D1/TBC1D1

0 p 0 -/- -/- -/- WTγ3 WT γ3 WT γ3 WT γ3-/- WT γ3-/-

Vehicle 991 MK-8722 Vehicle 991

M MK-8722 tolerance test 15 WT Vehicle γ3-/- WT MK-8722 10 γ3-/-

5 Blood glucose (mM)

0 0 50 100 150 Time (min) bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 6

A B )

BAT WT EDL WT -1 5 ∙mg

66 kDa -1 AMPKα1 4

AMPKα2 66 kDa ∙ min 3 AMPKα 66 kDa β1 2 AMPK β2 40 kDa 37 kDa

AMPKγ1 AMPK γ 3 Activity 1 GAPDH 40 kDa 0 (pmol Pi incorporated WT γ3-/- GAS WT BAT

C D IP: AMPKγ3 IP: AMPKγ1 GAS WT BAT WT GAS WT BAT WT IgG Sheep Post-IP IP Input Post-IP IP Input Post-IP IP Input IgG Rabbit Post-IP IP Input Post-IP IP Input Post-IP IP Input 75 kDa 75 kDa AMPKγ3 AMPKα1 75 kDa AMPKα1 75 kDa IB AMPKα2 75 kDa IB AMPKα2 β1 37 kDa AMPK β2 37 kDa β1 37 kDa AMPK β2 AMPKγ1

E BAT WT BAT γ3-/- 1.5 WT -/- AMPKα1 66 kDa γ3 AMPKα2 66 kDa 1.0 AMPKα 66 kDa

β1 AMPK β2 40 kDa 0.5 37 kDa AMPKγ1 (AMPK/GAPDH)

GAPDH 40 kDa Fold change relative to WT 0 α α1 α2 β1 γ1β2 AMPK subunits bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 7 A B C D -/- 3000 33 WT γ3 2500 WT * WT -/-

C) -/- γ3 ) * ° γ3 * * -1 * * 2000 · h

-1 2000 32 Saline μM) 1500 (ml · kg

2 1000 1000 31 NEFA ( VO 500 area temperature ( IS Top 10% mean surface

0 30 CL 316,243 0 e e 43 in 43 n 43 2 2 i 2 6, Saline Sal Sal 6, 31 31 CL 316, CL CL E FGH 4000 * WT 40 0.6 3 ) * * Saline WT -1 * -/- * * γ3 γ3-/- * * * · h 3000 * -1 WT 5D CL 30 γ3-/- 0.4 2 2000 20 * * (ml · kg 2 0.2 1 1000 Body weight (g)

VO 10 BAT weight (%BW) iWAT iWAT weight(%BW)

0 0 0.0 0 Basal Day 1 Day 2 Day 3 Day 4 Day 5 Saline 5D CL Saline 5D CL Saline 5D CL

I BAT J iWAT WT γ3-/- WT γ3-/- Saline Saline 5D CL 5D CL

K L M

4 30 1.5 40 WT Saline 5D CL WT -/- γ3 γ3-/- -/- -/- 3 30 WT γ3 WT γ3 * 1.0 * 20 * 37 kDa * UCP1 -tubulin 2 20 β Cidea Cox8b

MT-CO2 10 * 0.5 * β-Tubulin 1 10 50 kDa UCP1/ (Relative to WT Saline) (Relative to WT Saline) (Relative to WT Saline) (Relative to WT 5D CL) N.D. 0 0 0 0 Saline 5D CL Saline 5D CL Saline 5D CL Saline 5D CL bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Supplementary Figure 1 A Mouse genomic locus

5` UTR 1 2 3 4 5 6 7 8 9 10 11 12 13 3` UTR Targeting locus

Puro

1 2 3 4 5 6 7 8 9 10 11 12 Targeting allele LHA: 5.9 kb FR: 2.3 kb SHA: 3.2 kb

Puro

1 2 3 4 5 6 7 8 9 10 11 12 13

1 2 3 4 5 6 7 8 9 10 11 12 13 Conditional KO loxP site

FRT site

1 2 3 4 11 12 13 Constitutive KO

B GAS C WT γ3-/- Cumulative food intake

AMPK γ3 75 kDa 5

50 kDa 4

37 kDa 3

25 kDa 2

20 kDa Food intake (g) 1

15 kDa 0 10 kDa WT γ3-/- WT γ3-/- Dark Light

D E Total activity (motion) VO2 mean 20,000 5,000

4,000

15,000 (bw) -1

· g 3,000 10,000 -1 2,000 mL · h 5,000 Activity counts/h 1,000

0 0 WT γ3-/- WT γ3-/- WT γ3-/- WT γ3-/- Dark Light Dark Light bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Supplementary Figure 2 A B WT γ3+/- 1.5 WT γ3+/- 75 kDa AMPKγ3 1.0 AMPKα 66 kDa 66 kDa AMPKα2 # 0.5 β1 (AMPK/Vinculin) AMPK β2 40 kDa Fold change relative to WT Vinculin 116 kDa 0 α α2 β1 β2 γ3 AMPK subunits C D AMPKγ3 activity AMPKγ3 activity ) -1 60 * 4

∙mg * -1 * 3

∙ min 40 *# 2 20

Fold change 1 AMPK γ 3 activity reative to vehicle

0 0 (pmol Pi incorporated +/- +/- WT γ3 WT γ3 WT γ3+/- WT γ3+/-

Vehicle AICAR Vehicle AICAR 2 mM 2 mM E F G Ex vivo EDL 3 pS212-ACC pS231-TBC1D1 1.5 1.5 * * * * )

-1 *

h *

⋅ 2 1.0 1.0 -1 g ⋅

Mol 1 0.5 0.5 µ ( Glucose uptake pS212-ACC/ACC

0 0 pS231-TBC1D1/TBC1D1 0 WT γ3+/- WT γ3+/- WT γ3+/- WT γ3+/- WT γ3+/- WT γ3+/-

Vehicle AICAR Vehicle AICAR Vehicle AICAR

H Adenylate energy charge 1.0

0.8

0.6

0.4

0.5 ADP + ATP/ 0.2 ATP + ADP AMP

0 WT γ3-/- WT γ3-/- Vehicle AICAR bioRxiv preprint doi: https://doi.org/10.1101/2021.03.05.433354; this version posted March 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Supplementary Figure 3

MK-8722 tolerance test (30 mpk) 10 WT 8 γ3-/-

6

4

2 Blood Glucose (mM)

0 0 50 100 150 Time (min)