DOI: 10.5061/dryad.x95x69pkt
title: Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice

description: The loss of skeletal muscle function with age, known as sarcopenia,
 significantly reduces independence and quality of life and can have
 significant metabolic consequences. Although exercise is effective in
 treating sarcopenia it is not always a viable option clinically, and
 currently there are no pharmacological therapeutic interventions for
 sarcopenia. Here we show that chronic treatment with pan-adiponectin
 receptor agonist AdipoRon improved muscle function in male mice by a
 mechanism linked to skeletal muscle metabolism and tissue remodeling. In
 aged mice, 6 weeks of AdipoRon treatment improved skeletal muscle
 functional measures in vivo and ex vivo. Improvements were linked to
 changes in fiber type, including an enrichment of oxidative fibers, and an
 increase in mitochondrial activity. In young mice, 6 weeks of AdipoRon
 treatment improved contractile force and activated the energy sensing
 kinase AMPK and the mitochondrial regulator PGC-1a (peroxisome
 proliferator activated receptor gamma coactivator 1 alpha). In cultured
 cells, the AdipoRon induced stimulation of AMPK and PGC-1a was associated
 with increased mitochondrial membrane potential, reorganization of
 mitochondrial architecture, increased respiration, and increased ATP
 production. Furthermore, the ability of AdipoRon to stimulate AMPK and
 PGC1a was conserved in nonhuman primate cultured cells. These data show
 that AdipoRon is an effective agent for the prevention of sarcopenia in
 mice and indicate that its effects translate to primates, suggesting it
 may also be a suitable therapeutic for sarcopenia in clinical application.
description: <strong>Mice and Treatment:</strong> Two cohorts of
 mice were used. Six‐week‐old male B6C3F1 hybrid mice were obtained from
 Jackson Laboratories (Bar Harbor, ME, USA) and 24-month old male C57BL/6J
 mice from the NIA aged mouse colony. The animals were housed under
 controlled pathogen‐free conditions. The animals were fed ad-libitum
 Purina LabChow 5001 diet with free access to water, and placed on a 12h
 light/dark cycle. AdipoRon (10mg) (Adipogen, CA, USA) was reconstituted
 with 500ul of DMSO (stock) and stored at -20C. For the acute experiment,
 the animals were given either a single intravenous dose of AdipoRon @1.2
 mg/kg BW in PBS (n=5) or an equivalent volume of DMSO in PBS (n=3,
 Controls) in a total volume of 100ul. Extensor digitorum longus (EDL) and
 soleus muscle groups were harvested after 90 minutes. For the chronic
 experiments in young B6C3F1 mice (n=12) and old C57BL/6J mice (n=20), the
 animals in each age group were divided into 2 groups and received either
 AdipoRon @1.2 mg/kg BW in PBS or an equivalent volume of DMSO in PBS via
 tail vein injections three times per week (Monday, Wednesday and Friday)
 for 6 weeks. Body weight and food intake measurements were recorded once a
 week. Body composition was measured at the beginning and at the end of the
 treatment period using EchoMRI Body Composition Analyzer (Houston, TX). 
 At the end of the treatment period, animals were sacrificed. EDL, soleus,
 and gastrocnemius muscle specimens were mounted in OCT oriented to present
 fiber cross-section for tissue sectioning or snap frozen in liquid
 nitrogen and stored at -80°C. <strong>Fasting
 glucose and insulin measurements</strong>: Overnight fasting blood
 samples were collected for measures of glucose (One touch Ultra Blue
 glucometer), and insulin levels (Ultra-Sensitive Mouse Insulin ELISA Kit
 (CystalChem, IL, USA)). HOMA-IR (homeostasis model assessment of insulin
 resistance) index was calculated as [fasting serum glucose × fasting serum
 insulin/22.5].  <strong>Metabolic
 Chambers</strong>: Metabolic parameters and activity were measured
 (Columbus Instruments Oxymax/CLAMS metabolic chamber system (Columbus,
 OH)) in mice acclimated to housing in the chamber for approximately 24
 hours followed by 24hr continuous data collection period
 <strong>Ex vivo muscle force measurements</strong>:
 EDL and soleus hindlimb muscles were dissected and perfused with
 oxygenated (95% O2, 5% CO2) Tyrode’s solution at room temperature (NaCl
 145 mM, KCl 5 mM, CaCl2 2mM, MgCl2 0.5 mM, NaH2PO4 0.4 mM, NaHCO3 24 mM,
 EDTA 0.1 mM, Glucose 10 mM). Isolated muscles were attached to a
 contractile apparatus capable of measuring force (Aurora Scientific) and
 were electrically stimulated using parallel platinum electrodes. Maximal
 twitch force and tetanic force were determined by adjusting the length of
 the muscle until maximal twitch force is reached, defining the optimal
 length. A 15-minute period of rest allowed muscle to equilibrate to the
 new environment. Fatigability was defined as the decline in tetanic force
 following 10 minutes of continuous stimulation. Muscles were tetanically
 stimulated at 100 Hz for 500 ms every 5 seconds at a voltage that
 generates the maximal force. Following 10 minutes of fatiguing
 stimulation, a 20-minute recovery period was allowed, where there was no
 stimulation in an oxygenated Tyrode’s perfusion. After the recovery
 period, muscle was electrically stimulated again to quantify the amount of
 recovery force. A recovery force higher relative to fatiguing force was
 taken as an indication that the decline in force during fatiguing
 stimulation is reversible. The amount of fatigue and recovery force was
 represented as a percentage of the initial force (before fatiguing
 stimulation).  Following these measurements, fatigued muscles were
 weighed, quick frozen in liquid nitrogen and stored at -80°C. 
 <strong>Western blotting</strong>: Equal amounts of
 skeletal muscle protein extract (45µg) were separated on Mini-Protean TGX
 precast protein gels (Biorad, CA, USA) and transferred to a PVDF membrane
 using a Trans-Blot semi-dry transfer system (Biorad, CA, USA). The
 membranes were blocked using 5% BSA in TBST for phospho-antibodies or 5%
 non-fat milk for other antibodies for 1hr at RT. The following primary
 antibodies were used overnight: pAMPK Thr172 (Cell Signaling Technology
 #2535, Beverly, MA, USA), AMPK (Cell Signaling Technology #2532) and GAPDH
 (Cell Signaling Technology #2118), PGC1a (H300, Santa Cruz #sc-13067) and
 Beta-Actin (Sigma, #A1978), washed in TBST, and incubated with the
 respective HRP-conjugated secondary antibodies (Vector Labs, #PI:1000 and
 #PI:2000, Burlingame, CA, USA) for 1 hr at RT. Proteins were detected
 (Supersignal West Pico or Femto Chemiluminescent substrate solutions
 (Thermofisher Scientific)) and digital images acquired (GE ImageQuant Gel
 Doc Imaging system). Densitometric analysis was carried out using Fiji
 software. <strong>Real time PCR</strong>:
 RNA was extracted in Trizol reagent and isolated (Direct-zol RNA Miniprep
 kit (ZymoResearch, Irvine, CA, USA)). cDNA was synthesized from 1µg of RNA
 (High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems)). Real
 time PCR reactions were carried out using iTaq Universal SYBR Green mix
 (Biorad) with 10ng cDNA per reaction. The primer sequences are listed in
 Table S1 and the data was analyzed by 2^^ct method (Livak and Schmittgen,
 2001) and presented either as “relative expression” compared to 18S, or as
 “fold change” where relative expression of control samples were normalized
 to one. <strong>Histology</strong>: Serial
 OCT mounted cryostat sections (10 µm in thickness) were cut at -14ºC
 (Leica Cryostat (Fisher Supply, Waltham, MA)). Freshly cut sections were
 stained for cytochrome c oxidase activity and digital image captured the
 same day as described previously (Pugh et al., 2013). Briefly, sections
 were air-dried at RT for 10-15 minutes, incubated in a solution of 0.1M
 phosphate buffer, pH 7.6, 0.5 mg/ml DAB (3,3'-diaminobenzidine), 1
 mg/ml cytochrome c, and 2 µg/ml catalase at RT for ~30 minutes, washed
 with PBS, dehydrated, cleared and mounted under a glass coverslip
 (Permount (Fisher)). Similarly for succinate dehydrogenase activity
 staining, the frozen muscle sections were air-dried for 10-15 minutes and
 incubated in SDH reaction mixture containing [1.5mM nitroblue tetrazolium
 (NBT) and 50mM di-sodium succinate in 0.2M PBS (pH 7.6) at RT for ~20
 minutes, washed with PBS, dehydrated, cleared and mounted under a
 coverslip with Permount (Punsoni et al., 2017). Muscle fibrosis was
 determined by trichrome staining (Abcam, MA). For stain intensity and/or
 stain area analysis in digital images (n=5 per tissue), see
 below. <strong>Immunofluorescence</strong>:
 Fiber types (I, IIa, IIb and IIx) were quantified in gastrocnemius
 muscle.  Sections were air-dried for 30 minutes and then rehydrated with
 PBS for 5 minutes. Sections were then blocked with 0.5% BSA 0.5% Triton
 X-100 in PBS for 30 minutes at RT, then incubated with a cocktail of
 primary antibodies purchased from Developmental Studies Hybridoma Bank
 (DSHB, University of Iowa): BA-D5 (IgG2b, supernatant, 1:100 dilution)
 specific for MyHC-I, SC-71 (IgG1, supernatant, 1:100 dilution) specific
 for MyHC-IIa and BF-F3 (IgM, supernatant, 1:7.5 dilution) specific for
 MyHC-IIb for 1 hr at RT. After 3 washes with PBS (5 min each), the
 sections were incubated with secondary antibody cocktail (Invitrogen) to
 selectively bind to each primary antibody: goat anti-mouse IgG1 conjugated
 with Alexafluor488 (Invitrogen, # A21121); goat anti-mouse IgG2b
 conjugated with Alexafluor350 (Invitrogen, # A21140); goat anti-mouse IgM
 conjugated with Alexafluor594 (Invitrogen, # A211044) for 1 hr in the dark
 at RT. After 3 washes with PBS (5 min each) and a brief rinse in water,
 the sections were mounted in 85% glycerol in PBS for imaging. Type IIa
 fibers will appear green, IIb as red, I as blue and the fibers that are
 not stained by these antibodies will appear black and are classified as
 type IIx. <strong>Digital image capture and
 analysis</strong>: Digital images were captured using a 20x
 objective in a Leica DM4000B microscope equipped with Retiga 4000R digital
 camera (QImaging Systems, Surrey, BC, Canada). Background correction was
 conducted for all images using an unstained adjacent area of the slide.
 Images were converted to 8-bit format and inverted. Fiber type was
 classified based on immunostaining for myosin isoform and individual
 fibers were outlined using a free hand tool and the cross-sectional area.
 Stain intensity of the cytochrome c oxidase and succinate dehydrogenase
 was quantified using ImageJ/Fiji software. Using a custom-generated
 algorithm, the blue stained areas for collagen were separated and
 highlighted from the rest of the image and % stained area was quantified
 (MIPAR software). Mitochondrial analysis was performed on cells in ImageJ
 by applying image deconvolution, background subtraction, adaptive
 binarization, and segmentation algorithms followed by particle analysis
 and morphology analysis with the ImageJ plugin MiNA to quantify intensity
 and mitochondrial branching. <strong>Cell culture
 and reagents</strong>: NIH-3T3 fibroblasts were purchased (ATCC;
 CRL-1658) and cultured in DMEM supplemented with 10% bovine serum and 1%
 Penicillin/Streptomycin. The cells were treated with vehicle (DMSO in
 media) or AdipoRon (10µM or 50µM in media) for 10 minutes (AMPK
 phosphorylation) or 60 minutes (gene expression analysis). After
 treatment, cells were washed once with PBS and then lysed in Trizol or
 RIPA buffer depending on the experiment. C2C12 cells were purchased (ATCC;
 CRL-1772) and grown in 10% bovine serum and 1% penicillin/streptomycin.
 Differentiation was initiated after 2-3 days growth by exposure to
 differentiation media: Serum free DMEM supplemented with 2% equine serum,
 1% penicillin/streptomycin, and 172nM insulin. Myoblasts were fed
 differentiation media every 24h for 5-7 days until myotube morphology was
 reached. <strong>TMRE and Oxygen Consumption
 assays</strong>: Cells were treated with vehicle or AdipoRon (50µM)
 for 2 or 4 hrs. For TMRE fluorescence, cells were seeded at 5x104
 overnight and treated with AdipoRon (50um) for 2 or 4 hours. Cells then
 incubated for 30 minutes in 100nM TMRE dye in media and, after
 equilibrating for 10 minutes, plates were read at 530nm excitation/580nm
 emission, parallel treatment with 10 µM uncoupler FCCP (Cayman Chemical)
 accounted for mitochondrial oxidative phosphorylation. Data shown as
 fluorescent ratio of given treatment over uncoupled control.  For oxygen
 consumption assays, cells were trypsinized and equal numbers (4x105) were
 re-suspended in respiration buffer and loaded on to the PreSens Oxoplates
 (Regensburg, Germany). Appropriate ambient and anoxic controls were
 included in the same plate. <strong>ATP
 luminescence assay</strong>: Relative ATP levels were quantified
 (ATPLite™assay (Perkin-Elmer, Waltham, MA; INFINITE M1000 PRO microplate
 reader (TECAN, Grodig, Austria)) in cells cultured as above and treated
 with vehicle or AdipoRon (50µM) for the prescribed times.
 <strong>Cell Proliferation Assay</strong>: NIH-3T3
 cell proliferation was quantified according to the CyQUANT® Cell
 Proliferation Assay Kit (Molecular Probes, Inc. Eugene OR, USA) at four
 cell densities 1600 – 6700 cells/well. In brief, cells were seeded in
 microplate wells in growth medium at desired densities along with serial
 dilutions of cells for determination of a cell number standard curve.
 Cells were incubated for 24h after which culture medium was removed, and
 the number of cells quantified according to manufacturer
 instructions. <strong>Nonhuman Primate PBMC
 isolation and culture</strong>: Blood was collected from primates at
 the Wisconsin National Primate Research Center (WNPRC) with the approval
 of the Institutional Animal Care and Use Committee of the Office of the
 Vice Chancellor for Research and Graduate Education of the University of
 Wisconsin, Madison. PBMCs were isolated from 6ml of blood using SepMate-50
 tubes (Stemcell Technologies, Cambridge, MA, USA). Isolated primate PBMC’s
 (1x106) were cultured in 10cm plates in DMEM supplemented with 10% fetal
 bovine serum and 1% Penicillin/Streptomycin. Cells were treated with
 vehicle (DMSO in media) or AdipoRon (10µM or 50µM in media) for 5 minutes
 (AMPK phosphorylation) or 60 minutes (gene expression analysis). PBMCs
 were collected by centrifugation and the cell pellet washed and lysed in
 Trizol or RIPA buffer.