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Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice

Citation

Balasubramanian, Priya et al. (2022), Adiponectin receptor agonist AdipoRon improves skeletal muscle function in aged mice, Dryad, Dataset, https://doi.org/10.5061/dryad.x95x69pkt

Abstract

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.

Methods

Mice and Treatment: 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.

Fasting glucose and insulin measurements: 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]. 

Metabolic Chambers: 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

Ex vivo muscle force measurements: 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. 

Western blotting: 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.

Real time PCR: 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.

Histology: 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.

Immunofluorescence: 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.

Digital image capture and analysis: 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.

Cell culture and reagents: 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.

TMRE and Oxygen Consumption assays: 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.

ATP luminescence assay: 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.

Cell Proliferation Assay: 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.

Nonhuman Primate PBMC isolation and culture: 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.