The ex vivo model is a useful method for evaluating the impact of various physiological interventions and pharmacological compounds and muscle glucose uptake. This technique allows for the accurate assessment of glucose uptake in isolated mouse skeletal muscle in the absence of confounding factors that may interfere in an intact animal model. Before beginning an experiment, switch on the integrated muscle strip biograph system and warm the chambers to 30 degrees celsius.
Open the data collection software and calibrate the forced transducers to ensure comparability between data sets. Add four milliliters of pre-warmed 30 degree celsius basal incubation medium to each incubation chamber and make sure the medium is continuously oxygenated with 95%oxygen and 5%carbon dioxide. Place the mouse in the prone position on a dissection tray and confirm a lack of response to pedal reflex.
Then pin down a single front paw with a needle. Use scissors to remove the skin from the lower leg until both the achilles tendon and knee joint are visible. For dissection of the soleus muscle, attach a single 0.4 centimeter diameter suture loop prepared from a 16 centimeter non-absorbable surgical nylon piece of suture to the achilles tendon and secure peen forceps to the achilles tendon distally to the loop.
Cut to release the soleus and gastrocnemius muscles from the paw and carefully slide the peen forceps across the mouse to expose the soleus muscle. Pin down the peen forceps and place a second suture loop around the proximal tendon of the soleus muscle. Cut the proximal tendon and dissect the soleus including the two attached suture loops free of the gastrocnemius muscle.
Upon collection, use the loops to quickly attach the soleus muscle to the respective hooks in one of the incubation chambers. Use forceps to remove the fascia covering the tibialis anterior muscle. The distal tendons of the tibialis anterior or EDL muscles should be clear white, visible, and separated from each other.
Cut the distill tendon of the tibialis anterior muscle and dissect out the muscle. Use forceps to gently liberate the EDL muscle from the surrounding tissues, leaving the muscle intact without cutting the tendons. Place individual suture loops around the distill and proximal EDL tendons.
When the loops have been placed, cut the tendons to release the EDL muscle with the suture loops attached and quickly attach the loops to the hooks in the second incubation chamber. Then adjust the resting tension of each muscle to approximately five millinewtons and allow the muscles to equilibrate in the medium for at least 10 minutes before starting the experiment. To measure the insulin stimulated glucose uptake, replace the basal incubation medium in the chambers with the incubation medium without insulin, a submaximally effective insulin concentration, or a maximally effective insulin concentration for a 20 minute incubation.
At the end of the stimulation period, replace the incubation medium with glucose uptake incubation medium containing an identical concentration of insulin for a 10 minute incubation. At the end of the glucose uptake incubation, carefully remove the muscles from the incubation chambers and wash the samples in ice cold basal incubation medium. After the wash, quickly dry the muscles on filter paper and freeze the tissue without the suture loops in liquid nitrogen.
Then collect 100 microliters of the glucose uptake incubation medium from each chamber for 20 degree celsius storage and muscle glucose uptake analysis. To measure the contraction stimulated glucose uptake, place platinum electrodes centrally and on both sides of the muscles within the incubation chambers, and initiate muscle contraction immediately after replacing the basal incubation medium with glucose uptake incubation medium. Record the forced production from each incubated muscle.
After 10 minutes, gently collect and freeze the muscles as demonstrated, storing 100 microliter aliquots of the glucose uptake incubation medium from each chamber for muscle glucose uptake analysis as demonstrated. To determine the myocellular signaling by western blotting analysis in the same set of muscle samples, homogenize each frozen muscle sample in 400 microliters of ice cold homogenization buffer and rotate the sample end over end for one hour at four degrees celsius. Then collect the licate by centrifugalization and measure the amount of protein of interest within each sample pallet according to standard western blot analysis protocols.
To measure the glucose uptake in each sample, add 150 microliters of supernatant from each sample and 25 microliters of glucose uptake incubation medium from the corresponding incubation chambers to separate liquid scintillation counting vials containing three milliliters of liquid scintillation fluid per vial. Then load the vials onto a liquid scintillation counter and measure radioactivity of two deoxyglucose and mannitol according to the manufacturer's guidelines. In this representative analysis, the basal glucose uptake rates were similar between soleus and EDL muscles isolated from female mice.
Glucose uptake increased in soleus and EDL muscles in response to both submaximally and maximally effective insulin concentrations. Moreover, both submaximal and maximal insulin stimulated glucose uptakes were significantly higher in the soleus muscle. In contrast, contraction induced glucose uptake was significantly higher in the EDL compared to the soleus muscle.
Here, the maximal muscle force produced in soleus and EDL muscles during the 10 minute stimulation period can be observed. As expected, the EDL muscle generated more force during the initial part of the stimulation period, followed by a fast decline later in the stimulation period. Submaximal and maximal insulin concentrations induced an increase in the phosphorylation of Akt threonine 308 and TBC1D4 Serine 588, while contraction induced an increase in the phosphorylation of AMPK Alpha threonine 172 and ACC serine 212.
Neither insulin nor contraction led to a change in the total protein content. Before attempting to measure glucose uptake in isolated mouse muscles, it is vital to thoroughly practice the dissection of the soleus and EDL muscles. Following the assessment of the muscle glucose uptake, the remaining muscle protein lysate can be used for additional biochemical analysis that may elucidate the observed changes in glucose uptake rates.
