DOI: 10.5061/dryad.f1vhhmh96
title: Data from: Ampk alpha2 T172 activation dictates exercise performance and energy transduction in skeletal muscle

description: AMPK (5′-AMP-activated protein kinase) is an energetic sensor for
 metabolic regulation and integration. Here, we employed CRISPR/Cas9 to
 generate non-activatable Ampkα knock-in (KI) mice with a mutation of the
 threonine 172 phosphorylation site to alanine, circumventing the
 limitations of previous genetic interventions that disrupt the protein
 stoichiometry. KI mice of Ampkα2, but not Ampkα1, demonstrated phenotypic
 changes with increased fat-to-lean mass, impaired endurance exercise
 capacity, and diminished mitochondrial maximal respiration and conductance
 in skeletal muscle. Integrated temporal multi-omic analysis (proteomics/
 phosphoproteomics/ metabolomics) in skeletal muscle at rest and during
 exercise establishes a pleiotropic yet imperative role of Ampkα2 T172
 activation for glycolytic and oxidative metabolism, mitochondrial
 respiration, and contractile function. Importantly, there is a significant
 overlap of skeletal muscle proteomic changes in Ampkα2 T172A KI mice with
 those of type 2 diabetic patients. Our findings suggest that Ampkα2 T172
 activation is critical for exercise performance and energy transduction in
 skeletal muscle and may serve as a therapeutic target for type 2 diabetes.
description: <strong>LC-MS/MS Proteomics &
 Phosphoproteomics</strong> Muscle tissue samples
 from male and female WT and KI mice were collected before exercise (i.e.,
 sedentary control, SED), after 10-min running (10 min), and after
 exhaustion (Exh.) and stored at -80ºC until undergoing processing. Sample
 processing for global proteomics and phosphoproteomics analysis was
 conducted as previously described (<em>71</em>) with several
 modifications. Briefly, frozen muscle tissue samples were minced into
 small pieces on a pre-chilled aluminum tray with dry ice. ~ 25 mg of
 tissue were collected and homogenized in cold lysis buffer (50 mM Tris, 8
 M urea, 75 mM NaCl, 1 mM EDTA, 2 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM
 PMSF, 10 mM NaF, 1% phosphatase inhibitor cocktail 2, 1% phosphatase
 inhibitor cocktail 3, pH 8.0) on a pre-chilled bead beater using 2-min
 cycle. Samples were then centrifuged at 13,000xg at 4 °C for 10 min to
 remove debris. The extracted protein was quantified by the CA assay. The
 same quantity of protein was aliquoted and reduced with 5 mM DTT for 1
 Ampk shaking on a thermomixer at 37 °C, 1000 rpm. Proteins were then
 alkylated with 20 mM iodoacetamide with shaking in the dark at room
 temperature, 1000 rpm for 45 min. Samples were diluted 4-fold, then
 digested with trypsin (enzyme to protein ratio = 1: 50) for 1 hour,
 shaking at 1000 rpm, 37°C. A fresh aliquot of trypsin (enzyme to protein
 ratio = 1: 50) was added after the samples were diluted 2-fold. Then the
 digestion was conducted at 37°C, 1,000 rpm overnight. The digest was
 desalted by Sep-Pak C18 SPE cartridges (Waters, Milford, MA). Clean
 peptides were quantified by the BCA assay. 250 µg of peptides from each
 sample was aliquoted and concentrated in a speedvac to completely dry, to
 be used for TMT labeling. Peptides were resuspended in
 500 mM HEPES (pH 8.5) at 5 μg/μL. Three replicates of male or female
 muscle tissue samples from each condition (WT-SED, WT-10 min, WT-Exh.,
 KI-SED, KI-10 min, KI-Exh.) were labeled with two TMT18 plexes. TMT
 reagents were resuspended in anhydrous acetonitrile at 20 μg/μL and added
 to each sample at a 1:2.5 (peptide: TMT) ratio. Labeling was conducted at
 25 °C, 850 rpm for1 hourr shaking on a thermomixer. Then the reaction was
 quenched by hydroxylamine. Samples from each plex were combined and
 concentrated in a SpeedVac,c followed by C18 SPE cleanup. The clean
 TMT-labeled samples were then fractionated into 12 fractions using high pH
 reversed-phase separation. 5% of each fraction was used for global
 proteomics analysis; the remaining fractions were subjected to immobilized
 metal affinity chromatography (IMAC) phosphoenrichment using freshly
 prepared Fe<sup>3+</sup>-NTA-agarose beads.
 Both global and phosphopeptide fractions were analyzed usinga 
 Waters nanoAcquity UHPLC system (with 20 cm x 75 um i.d. 1.9-um column
 packed in-house with Waters BEH C18 silica) coupled to an Orbitrap Fusion
 Lumos (Thermo Scientific, San Jose, CA) with a 120-min LC gradient.
 Positive ion mode spray voltage was set at 2.2 kV. Full MS spectra were
 recorded at a resolution of 60K with a scan range of 350 – 1800 m/z.
 Automated gain control (AGC) value was set 4to e5. MS/MS was acquired in
 data-dependent mode (DDA) at a resolution of 50K, AGC of 1e5. Isolation
 window was 0.7 m/z. High-energy collision dissociation (HCD) with a
 normalized collision energy setting of 30% was used. Dynamic exclusion
 time was set at 45s. <strong>Untargeted
 Metabolomics</strong> Muscle tissue samples from
 male and female wild-type (WT) and knock-in (KI) mice were collected as
 described above. For extraction, tissues were thawed on ice, and
 approximately 750 µL of a cold chloroform: methanol (1:2) mixture was
 added, along with four steel balls (Fisher Brand; diameter 2.4 mm). Tubes
 were plunged into liquid nitrogen for 5 minutes and vigorously shaken in a
 Fisher Brand Bead Mill 24. Tubes were then vortexed and shaken at 900 rpm
 for 30 minutes at 4°C in a temperature-controlled thermal shaker. After
 adding 400 µL of water, samples were vortexed, and the upper aqueous phase
 was recovered as the metabolite mixture. Two hundred microliters of the
 extract were dried overnight in a SpeedVac and reconstituted in 200 µL of
 0.1% formic acid (FA) containing the 100X Metabolomics QReSS™ Kit
 (Cambridge Isotopes, MSK-QRESS-KIT). A 10 µL aliquot from each tube was
 removed to create a pooled quality control (QC) sample, which was injected
 at the beginning and end of the mass spectrometry (MS) sequence.
 Additional QC samples were injected after every five sample
 injections. LC-MS data acquisition was performed using
 a fully automated AcquireX Intelligent Data Acquisition Workflow with
 additional instrument parameters. The data acquisition and data processing
 workflow is based on a previous publication (<em>72</em>).  We
 first generated an exclusion list from a reagent blank sample to determine
 the background. A pooled quality control (QC) sample was injected for
 feature detection and component assembly to create an inclusion list.
 Using the inclusion list, a series of iterative data-dependent acquisition
 (DDA) injections were performed, with each injection informed by the
 previous one. Precursors from the inclusion list were fragmented, and once
 detected, they were automatically transferred to the exclusion list. This
 approach minimized redundant fragmentation and maximized relevant spectra
 and metabolite annotation. Samples were analyzed on a Thermo Orbitrap IDX
 Tribrid MS coupled to a Thermo Vanquish UHPLC. Metabolite separation was
 achieved using a Waters BEH C18 column (Waters Corp.; 2.1 × 150 mm, 1.7
 µm) maintained at 30°C. Samples were analyzed in both positive and
 negative ionization modes   <strong>AMPK
 Consensus Motif Mapping</strong> Each peptide was
 15 amino acids in length, centered on the phosphorylated residue (position
 0). Motif detection was performed based on known AMPK substrate
 preferences (<em>52</em>). Three levels of stringency were
 applied: strict, relaxed, and minimal motif match. The pattern for strict
 match was [I/V/L/M] - X - [R/K] - X - X - [S/T] - X - X - X - [I/F/L/M],
 in which the −5 position must be I, V, L, or M, −3 must be R or K, and +4
 must be I, F, L, or M. The pattern for relaxed match was X - X - [R/K] - X
 - X - [S/T] - X - X - X - [I/F/L/M]. For broader detection, a minimal
 motif was annotated requiring the presence of a basic residue at -3 with
 the pattern of X - X - [R/K] - X - X - [S/T] - X - X - X - X -
 X.
description: # Data from: Ampk alpha2 T172 activation dictates exercise performance and
 energy transduction in skeletal muscle Dataset DOI:
 [10.5061/dryad.f1vhhmh96](https://doi.org/10.5061/dryad.f1vhhmh96) ##
 Description of the data and file structure Muscle tissues from male and
 female WT and KI mice were collected at rest (SED), after 10 min of
 running, and at exhaustion, snap-frozen, and stored at −80 °C. Samples
 were processed for global proteomics and phosphoproteomics with minor
 modifications to established methods, including tissue homogenization,
 protein extraction, digestion, TMT18 labeling, high-pH fractionation, IMAC
 phosphoenrichment, and LC–MS/MS analysis on an Orbitrap Fusion Lumos.
 Untargeted metabolomics was performed using chloroform–methanol
 extraction, AcquireX-enabled LC–MS/MS on an Orbitrap IDX Tribrid, and
 rigorous QC procedures. AMPK consensus motifs were identified from
 phosphopeptides using strict, relaxed, and minimal sequence criteria based
 on known substrate preferences. ### Files and variables **File:
 S1_AMPK_global_DA_CAMERA_male_and_female.xlsx** **Description:**
 Differential abundance analysis of global proteomics data from
 gastrocnemius muscle of male and female mice. Results summarize pathway-
 and gene-level responses across experimental contrasts. **Variables:**
 Contrast (comparison being tested), GeneSetID and GeneSet (pathway or
 functional annotation), Direction (overall regulation), ZScore (gene set
 enrichment score), PAdj (adjusted p-value), gene (gene symbol), protein
 (protein identifier), description (protein annotation), logFC (log fold
 change), zscore (gene-level z-score), adj.P.Val (adjusted p-value).
 **File: S2_AMPK_phospho_DA_male_and_female_w_delta10min_deltaExh_XL.xlsx**
 **Description:** Differential abundance results from phosphoproteomics of
 gastrocnemius muscle in male and female mice, including responses to
 10-min exercise and exhaustion. **Variables:** contrast (comparison),
 featureName (phosphopeptide identifier), uniprot_acc (UniProt accession),
 entry_name (UniProt entry), site (phosphorylation site), sequence_window
 (local peptide sequence), description (protein annotation), gene (gene
 symbol), logFC (log fold change), AveExpr (average expression), t
 (t-statistic), P.Value (raw p-value), adj.P.Val (adjusted p-value),
 df.total (degrees of freedom), zscore (standardized effect size), type
 (phosphorylation category). **File: S3_AMPK_phosphoprot_IPA_results.xlsx**
 **Description:** Ingenuity Pathway Analysis (IPA) results derived from the
 phosphoproteomics data of male and female gastrocnemius muscle. Each entry
 corresponds to an IPA output generated from datasets listed in
 Supplemental File 2. **Variables:** filename (source dataset), set (IPA
 pathway or function), logp (−log10 p-value), ratio (proportion of pathway
 genes represented), z (activation z-score), genes (mapped genes), sex,
 group, timepoint, pval (raw p-value), contrast (comparison), padj
 (adjusted p-value). **File: S4_AMPK_consensus_motif_target_results.xls**
 **Description:** Identification of AMPK consensus motif targets from
 phosphoproteomics data in male and female gastrocnemius muscle. Motif
 matches are classified by stringency. **Variables:** Protein (protein
 name), Site (phosphorylation site), Sequence (peptide sequence), minus5,
 minus3, center, plus4 (key motif positions), strict_match, relaxed_match,
 Mininal_Motif (motif match categories). **File:
 S5_AMPK_metab_DA_male_and_female.xlsx** **Description:** Differential
 abundance analysis of untargeted metabolomics data from gastrocnemius
 muscle of male and female mice across experimental contrasts.
 **Variables:** contrast (comparison), featureName (metabolite feature ID),
 input_name (original annotation), standardized_name (harmonized metabolite
 name), super_class, main_class, sub_class (metabolite classifications),
 logFC (log fold change), AveExpr (average abundance), t (t-statistic),
 P.Value (raw p-value), adj.P.Val (adjusted p-value), df.total (degrees of
 freedom), zscore (standardized effect size). #### Code/software Any
 program that will open a spreadsheet, such as Excel, is recommended. ####
 Access information Other publicly accessible locations of the data: * None
 Data was derived from the following sources: * None