Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Summary

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Abstract

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.

Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [³H]2-deoxy-D-glucose and [¹⁴C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Introduction

Skeletal muscle possesses the ability to extract large quantities of glucose from the extracellular space in response to insulin and exercise. This helps to maintain whole-body glucose homeostasis and secures glucose supply during times of high energy demand. Since intact regulation of skeletal muscle glucose uptake has been shown to be important for overall health and physical performance^1,2, measurements of muscle glucose uptake during various conditions have received much attention. In humans and animals, the hyperinsulinemic-euglycemic clamp has been used as the gold standard technique to assess insulin sensitivity in vivo^3,4. In contrast to findings obtained from an oral glucose tolerance test, the hyperinsulinemic-euglycemic clamp technique does not require intact gastrointestinal function or insulin secretion from the pancreas and thus permits insulin responses to be compared between subjects who exhibit variations in gastro-intestinal and/or pancreatic function. Measurements of muscle glucose uptake in vivo during exercise in humans have been performed frequently since the 1960s⁵. First by the use of arteriovenous balance techniques⁶ and later by the use of positron emission tomography (PET) imaging in combination with a positron emitting glucose analogue e.g. ¹⁸F-Fluoro-deoxy-glucose⁷. In rodents, exercise-stimulated muscle glucose uptake in vivo is typically performed by the use of radioactive or stable isotope-labeled glucose analogues^8,9,10.

A complementary method to measurements of muscle glucose uptake in vivo, is to isolate and incubate small muscles from rodents and subsequently measure glucose uptake using radioactive or stable isotope-labeled glucose analogues^11,12,13. This method allows accurate and reliable quantification of glucose uptake rates in mature skeletal muscle and can be performed in the presence of various insulin concentrations and during contraction elicited by electrical stimulation. More importantly, measurements of glucose uptake in isolated and incubated skeletal muscle are of relevance when investigating the muscle metabolic phenotype of mice that have undergone various interventions (e.g. nutrition, physical activity, infection, therapeutics). The isolated skeletal muscle model is also a suitable tool for pharmacological compound testing that may affect glucose uptake per se and/or modify insulin sensitivity^12,14. In this way, the efficacy of compounds designed to regulate muscle glucose metabolism can be tested and evaluated in a highly controlled milieu before subsequent in vivo testing in pre-clinical animal models.

Under some conditions, metabolic viability may pose a challenge in the isolated and incubated skeletal muscle model system. Indeed, the lack of a circulatory system in the incubated muscles entails that delivery of substrates (e.g. oxygen and nutrients) fully depends on simple diffusion between the muscle fibers and the surrounding environment. In regards to this, it is of importance that the incubated muscles are small and thin and thus, represent less of a barrier for oxygen diffusion during incubation¹⁵. Especially during prolonged incubations for several hours, hypoxic states may develop due to insufficient oxygen supply resulting in muscle energy depletion¹⁵. Although various markers of metabolic viability in incubated rat muscle have been reported previously alongside the identification of important variables that help to maintain rat muscle viability¹⁵, a comprehensive evaluation of metabolic viability in small incubated mouse muscles is still warranted. Hence, at present, glycogen content has mainly been used as a marker of metabolic viability in incubated mouse skeletal muscle^16,17.

Here we describe a detailed protocol to measure basal, insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle from mice using radiolabeled [³H]2-deoxy-D-glucose and [¹⁴C]mannitol as an extracellular marker. In the present study, glucose uptake was measured during a 10-minute period and the method is presented with the use of submaximally and maximally effective insulin concentrations as well as a single contraction protocol. However, the protocols described herein can easily be modified with regards to incubation time, insulin-dosage, and electrical stimulation protocol. Furthermore, we provide a thorough characterization of various markers of metabolic viability in incubated soleus and EDL mouse muscle. The results indicate that glucose supplementation to the incubation buffer is essential to preserve metabolic viability of muscle incubated for 1 hour.

Protocol

Procedures involving research animals should be performed in accordance with relevant guidelines and local legislation. All animal experiments used for this study complied with the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes and were approved by the Danish Animal Experiments Inspectorate. 

1. Preparation of the experimental apparatus and suture loops

NOTE: For this study, use an integrated muscle strip myograph system with customized incubation hooks to incubate isolated mouse skeletal muscles (Figure 1). This system allows muscle to bathe in a physiological solution with continuous oxygenation (95% O₂ and 5% CO₂) and at constant temperature. The muscle tissue bath is coupled to a force transducer for the measurement of muscle force production during contraction. To elicit and record myo-mechanical responses during contraction, employ an electrical pulse stimulator and a data collection program, respectively. Stimulate the incubated muscles to contract by platinum electrodes positioned centrally and on both sides of the muscle.

1. Turn on the myograph system and warm chambers to 30 °C. Open data collection software compatible with the myograph system and calibrate force transducers to ensure comparability between datasets.
2. Start by cutting ~16 cm strands of non-absorbable surgical nylon suture. Use forceps to create a loop of approximately 0.4 cm in diameter from a single strand. Repeat this until enough loops have been produced. Each muscle needs two loops - one for the proximal and one for the distal tendon.

2. Preparation of solutions and incubation media

1. Preparation of basal incubation media
   1. Prepare the following stock solutions: 2.5 M sodium chloride (NaCl, 250 mL), 0.5 M sodium bicarbonate (NaHCO₃, 250 mL), 0.5 M potassium chloride (KCl, 50 mL), 0.25 M calcium chloride (CaCl₂, 50 mL), 0.25 M potassium dihydrogen phosphate (KH₂PO₄, 50 mL), 0.25 M magnesium sulfate (MgSO₄, 50 mL), 110 mM sodium pyruvate (Na-Pyruvate, 100 mL), 500 mM D-mannitol (100 mL), 1 M 2-deoxy-D-glucose (4 mL), 15% solution of bovine serum albumin (BSA) dialyzed against Krebs-Ringer-Henseleit (KRH) buffer (described below in step 4) (100 mL).
      ​NOTE: Two solutions are required to measure resting and contraction-stimulated glucose uptake. Furthermore, each single insulin concentration used to assess insulin-stimulated glucose uptake requires two solutions. Thus, in total six different solutions are needed to measure basal, submaximal insulin-, maximal insulin-, and contraction-stimulated glucose uptake in isolated mouse skeletal muscle. In the following, 'basal incubation media' refers to media without insulin or radioactive tracers. 'Incubation media' refers to media containing insulin. 'Glucose uptake incubation media' refers to media containing 2-deoxy-D-glucose and radioactive tracers in addition to insulin at a concentration identical to that used in the 'incubation media'.
   2. Prepare a KRH buffer by supplementing ultrapure water (ddH₂O) with NaCl (117 mM), NaHCO₃ (24.6 mM), KCl (4.7 mM), CaCl₂ (2.5 mM), KH₂PO₄ (1.2 mM), and MgSO₄ (1.2 mM). Subsequently, gas the KRH buffer with 95% O₂ and 5% CO₂ for at least 10 min. The desired pH of the KRH buffer should be between 7.35-7.45 at 30 °C. If pH adjustment is performed at room temperature the pH of the KRH buffer should be between 7.25-7.35.
   3. Add BSA (0.1%), Na-Pyruvate (2 mM), and D-mannitol (8 mM) to the gassed and pH-adjusted KRH buffer to complete the basal incubation media. Store basal incubation media in a sealed container to minimize degasification of O₂ and CO₂ and place media at 30 °C.
      NOTE: Typically, the osmolarity of the KRH supplements (i.e. Na-Pyruvate, D-Mannitol, and D-glucose) is kept constant across an entire experiment to avoid shrinkage or expansion of the muscle cells. The protocol described herein uses an osmolarity of 10 mM for the KRH supplements. If a glucose-containing buffer is needed, replace KRH supplements to accommodate the needs, e.g. 5 mM D-glucose and 5 mM D-mannitol.
   4. To avoid possible BSA-associated contaminants in the incubation buffer, dialyze BSA against KRH.
      1. To make a 15% BSA stock solution dialyzed against KRH buffer, start by dissolving 300 g of analytical grade fat-free BSA in 900 mL of KRH buffer. Next, boil the dialysis tube in redistilled water until the tubing is soft.
      2. Fill the tubing with the BSA-KRH solution and secure the tubing ends. Place the tubing with BSA-KRH in 5 L of KRH buffer and leave it overnight at 4 °C. The following day replace the KRH buffer and leave the tubing with BSA-KRH in KRH buffer overnight at 4 °C.
      3. Lastly, collect the BSA-KRH solution from the tubing and add KRH buffer to a final volume of 2 L (i.e., 15% BSA-KRH stock solution). Divide the 15% BSA-KRH stock solution into aliquots and store in freezer at ~-20 °C.
2. Preparation of incubation media containing insulin
   1. For the incubation media containing a submaximally effective insulin concentration, add 1 µL of a 100 mU/mL insulin stock solution per mL of basal incubation media (100 µU/mL insulin).
   2. For the incubation media containing a maximally effective insulin concentration, add 1 µL of a 10 U/mL insulin stock solution per mL of basal incubation media (10 mU/mL insulin).
3. Preparation of glucose uptake incubation media
   ​​CAUTION: Handling of radioactive material is only allowed in a restricted and controlled area by authorized personnel and some universities, research institutions and companies may require the acquisition of a "Radioactivity Use Permit". Material and waste must be handled according to appropriate local procedures, guidelines, and legislation.
   1. Follow the same procedure as described in section 2.1.2.
   2. Add BSA (0.1%), Na-Pyruvate (2 mM), D-mannitol (7 mM), and 2-deoxy-D-glucose (1 mM) to the gassed and pH-adjusted KRH buffer.
   3. Add [³H]2-deoxy-D-glucose (0.028 MBq/mL) and [¹⁴C]mannitol (0.0083 MBq/mL) to the supplemented KRH buffer to complete the glucose uptake incubation media. Store at 30 °C. If [³H]2-deoxy-D-glucose and [¹⁴C]mannitol are dissolved in ethanol remove the ethanol by N₂ mediated evaporation before use.
   4. For the glucose uptake incubation media containing a submaximally effective insulin concentration, add 1 µL of a 100 mU/mL insulin stock solution per mL of glucose uptake incubation media (100 µU/mL insulin).
   5. For the glucose uptake incubation media containing a maximally effective insulin concentration, add 1 µL of a 10 U/mL insulin stock solution per mL of glucose uptake incubation media (10 mU/mL insulin).

3. Animals and dissection of the mouse soleus and EDL muscle for incubation

NOTE: Procedures involving research animals should be performed in accordance with relevant guidelines and local legislation. The described procedure can be used with in-house bred or commercially available male and female mice of various strains and genetic backgrounds. The following procedure is provided for fed female C57Bl/6J mice. On average, mice were 19 weeks old and weighed 25 g. The mice were maintained on a 12:12 h light-dark cycle with free access to standard rodent chow and water. Animal experiments were initiated at ~ 9:00 AM local time and all animals were sacrificed within a period of 2 h.

1. Add 4 mL of pre-warmed (30°C) basal incubation media to each incubation chamber and make sure the basal incubation media is continuously oxygenated with 95% O₂ and 5% CO₂.
2. Anaesthetize mice with an intraperitoneal injection of pentobarbital (10 mg/100 g body weight) or other available anaesthesia (e.g. tribromoethanol).
   ​NOTE: Be aware that in some countries a licence to handle pentobarbital and other anaesthetic drugs may be required. Before muscle dissection can be initiated, anaesthesia of each animal must be properly induced. To ensure this, tail and leg reflexes are tested. For optimal results dissection should be well practiced to avoid damaging the muscles during removal.
3. Place anaesthetized mice prone on a dissection tray (e.g. styrofoam lid) and pin down front and hind paws, as necessary, using a needle.
4. Remove the skin from the lower leg and make sure that both the Achilles tendon and knee joint are visible.
   1. For the dissection of soleus muscle, start by attaching a single suture loop to the Achilles tendon. Secure a pean forceps to the Achilles tendon distally of the suture loop and cut to release the soleus and gastrocnemius muscles from the paw. Carefully slide the pean forceps across the mouse thereby exposing the soleus muscle.
   2. Pin down the pean forceps and place a second suture loop around the proximal tendon of the soleus muscle. Next, cut the proximal tendon and dissect soleus (including the two attached suture loops) free of gastrocnemius muscle. Quickly place the soleus muscle in the incubation chamber by attaching each suture loop to the respective hooks.
5. Remove the fascia covering the m. tibialis anterior (TA) using forceps. If done correctly, distal tendons of the TA and EDL muscles should be clear white and visible; and separated from each other.
6. Cut the distal tendon of the TA muscle and dissect out the muscle for later analyses (e.g. genotyping). Using forceps, gently liberate the EDL muscle from the surrounding tissues but leave the muscle intact and do not cut the tendons. Place one suture loop around the distal tendon and a second suture loop around the proximal tendon of EDL.
7. Next, cut the tendons releasing the EDL muscle with two attached suture loops and quickly place the muscle in the incubation chamber by attaching each suture loop to the respective hooks. In order to not lose tension during incubation and especially during electrically-induced contraction of the soleus and EDL muscles, it is of great importance to fix the suture loops around the tendons with tight knots.
8. Lastly, euthanize the animal by e.g. cervical dislocation.
9. When the muscles have been dissected and placed in incubation chambers, adjust the resting tension of each muscle to ~5 mN and pre-incubate the muscles for at least 10 min before initiating the experimental protocol.

4. Insulin-stimulated glucose uptake in isolated mouse skeletal muscle

1. Following step 3.9 replace the basal incubation media with incubation media containing no insulin (basal incubation media), a submaximally effective insulin concentration or a maximally effective insulin concentration and leave in the incubation chambers for 20 min. Space each incubation chamber by 1 min, thereby making time for the subsequent harvest of muscles.
2. At the end of the 20 min stimulation period, replace the incubation media with the glucose uptake incubation media containing an identical concentration of insulin and leave in the incubation chambers for 10 min, again with 1 min spacing between each incubation chamber.
3. After 10 min of incubation in the glucose uptake incubation media gently remove muscles from the incubation chambers and wash them in ice-cold basal incubation media. Subsequently, quickly dry the muscles on filter paper before the suture loops are removed and muscles are frozen in liquid nitrogen. It is imperative that the incubated muscles are harvested quickly if one also wishes to investigate various intracellular metabolites and protein signaling in addition to glucose uptake.
4. Collect 100 µL of the glucose uptake incubation media from each incubation chamber and store it at -20 °C. The amount of radioactivity in these samples will be included in the calculation of muscle glucose uptake.

5. Contraction-stimulated glucose uptake in isolated mouse skeletal muscle

NOTE: To induce contraction of isolated mouse skeletal muscle use the following protocol: 1 train/15 s, each train 1 s long consisting of 0.2 ms pulses delivered at 100 Hz. However, other similar protocols eliciting contraction of isolated mouse skeletal muscle will likely work as well. Importantly, the voltage should be adjusted to generate maximal force development of the incubated muscle, which is dependent on the experimental setup. If this is not ensured, you may risk that not all fibers of the muscle are contracting. In turn, this may induce bias in the dataset.

1. Following step 3.9 place the platinum electrodes centrally and on both sides of the muscles. Initiate contraction of the muscles immediately after replacing the basal incubation media with the glucose uptake incubation media. If possible, space each incubation chamber by 1 min, thereby making time for the subsequent harvest of muscles. Remember to record force production from each incubated muscle.
2. After 10 min of contraction in the glucose uptake incubation media, remove the platinum electrodes, gently collect the muscles from the incubation chambers and wash them in ice-cold basal incubation media. Subsequently, quickly dry the muscles on filter paper before the suture loops are removed and muscles frozen in liquid nitrogen. The entire muscle harvest procedure should be performed as fast as possible.
3. Collect 100 µL of the glucose uptake incubation media from each incubation chamber and store it at -20 °C. The amount of radioactivity in these samples will be included in the calculation of muscle glucose uptake.

6. Skeletal muscle homogenization and processing

NOTE: The procedure given below for muscle homogenization makes it possible to determine both glucose uptake and myocellular signaling by western blotting in the same set of muscle samples.

1. Homogenize each muscle in 400 µL of ice-cold buffer with pH 7.5 containing 10% glycerol, 20 mM sodium-pyrophosphate, 1% IGEPAL CA-630 (NP-40), 2 mM phenylmethylsulfonylfluoride (dissolved in isopropanol), 150 mM NaCl, 50 mM HEPES, 20 mM β-glycerophosphate, 10 mM sodium fluoride (NaF), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM glycoletherdiaminetetraacetic acid (EGTA), 10 µg/ml aprotinin, 10 µg/mL leupeptin, 3 mM benzamidine, and 2mM sodium-orthovanadate using steel beads and a tissuelyser (2 x 45 s at 30 Hz). Rotate all homogenates end-over-end for 1 h at 4 °C after which they are centrifuged at 16,000 x g for 20 min at 4 °C. Collect the lysate (supernatant) which is used to determine muscle glucose uptake.

7. Determination of radiolabeled 2-deoxyglucose and mannitol

1. Add 150 µL of each muscle lysate and 25 µL of the glucose uptake incubation media from each incubation chamber to separate liquid scintillation counting vials containing 2 mL of liquid scintillation fluid. Moreover, prepare two blind control vials only containing 3 mL of liquid scintillation fluid. Close all the vials and mix thoroughly by vortexing each vial for ~5 s.
2. Place the vials in a liquid scintillation counter and measure radioactivity of [³H]2-deoxy-D-glucose and [¹⁴C]mannitol according to the manufacturer's guidelines. Record DPM (disintegrations per minute) for each liquid scintillation vial.

8. Calculation of muscle glucose uptake rates

1. Use the lysate from step 6.1 to measure the total protein concentration in each muscle sample using standard protein quantification methods (e.g., Bicinchoninic acid or Bradford assays). Calculate the amount of protein (mg) added to each scintillation vial.
   NOTE: The rate of glucose uptake for each muscle sample is calculated by subtracting the amount of [³H]2-deoxy-D-glucose located in the extracellular space from the total amount of [³H]2-deoxy-D-glucose in the muscle sample using [¹⁴C]mannitol as an extracellular marker. It is assumed that [³H]2-deoxy-D-glucose and [¹⁴C]mannitol exhibit similar diffusion properties within the muscle tissue during incubation. Perform the following calculations:
2. Start by subtracting the [³H] and [¹⁴C] DPM of the blind control samples from all muscle and media samples.
3. Determine the muscle extracellular space in µL (µL-ECS):
   [¹⁴C]DPM_muscle / ([¹⁴C]DPM_media / M_vol)
4. Calculate the amount of [³H]DPM in the muscle extracellular space ([³H]DPM_ECS):
   µL-ECS × ([³H]DPM_media / M_vol)
5. Calculate the amount of [³H]DPM in the muscle intracellular space ([³H]DPM_ICS):
   [³H]DPM_muscle− [³H]DPM_ECS
6. Calculate the muscle glucose uptake rate (µmol / g protein / hour):
   [³H]DPM_ICS / ([³H]DPM_media / M_vol) / [2-deoxy-D-glucose])) / mg protein) / T_h
   NOTE: For all the equations above,
   [¹⁴C]DPM_muscle is the amount of [¹⁴C]mannitol radioactivity in a muscle sample;
   [¹⁴C]DPM_media is the amount of [¹⁴C]mannitol radioactivity in a media sample;
   [³H]DPM_muscle is the amount of [³H]2-deoxy-D-glucose radioactivity in a muscle sample;
   [³H]DPM_mediais the amount of [³H]2-deoxy-D-glucose radioactivity in a media sample;
   [³H]DPM_ECS is the amount of [³H]2-deoxy-D-glucose radioactivity in the muscle extracellular space;
   [³H]DPM_ICS is the amount of [³H]2-deoxy-D-glucose radioactivity in the muscle intracellular space;
   µL-ECS is the muscle extracellular space in µL;
   M_vol is the volume (µL) of incubation media used for scintillation counting (e.g. '25' as mentioned above);
   T_h is the time factor used to calculate uptake rates per hour (i.e., '1/6' when incubating muscles with glucose uptake media for 10 min)
7. Take into consideration this example calculation. The [³H] and [¹⁴C] DPM of the blind control samples (17 and 6, respectively) have been subtracted from the DPM values mentioned below.
   [¹⁴C]DPM_muscle: 343
   [¹⁴C]DPM_media: 11846
   [³H]DPM_muscle: 4467
   [³H]DPM_media: 39814
   M_vol: 25
   mg protein: 0.396 (in 150 µL muscle protein lysate)
   [2-deoxy-D-glucose]: 1 (mM)
   T_h: 1/6 (h)
   µL-ECS = 343 DPM / (11846 DPM / 25 µL) = 0.724 µL
   [³H]DPM_ECS: 0.724 µL × (39814 DPM / 25 µL) = 1153 DPM
   [³H]DPM_ICS: 4467 DPM - 1153 DPM = 3314 DPM
   Glucose uptake: ((3314 DPM / (39814 DPM / 25 µL) / 1 mmol/L) / 0.396 mg protein) / (1/6 hour) = 31.53 µmol / g protein / hour

9. SDS-PAGE and western blot analyses

1. Prepare soleus and EDL muscle lysates in Laemmli buffer and heat for 5 min at 96 °C.
2. Separate equal amounts of muscle protein by SDS-PAGE on self-cast gels and transfer the proteins to polyvinylidene fluoride membranes by semidry blotting.
3. Subsequently, incubate membranes in Tris-buffered saline containing 0.05% Tween 20 and 2% skim milk and probe membranes with relevant primary and secondary antibodies.
4. Detect proteins with chemiluminescence and visualize them by a digital imaging system.

10. Muscle glycogen, nucleotides, lactate, creatine, and phosphocreatine

1. Use perchloric acid to extract EDL and soleus muscle samples.
2. Subsequently, neutralize samples and analyze them for lactate, creatine, and phosphocreatine as previously described¹⁸.
3. Analyze nucleotide content in EDL and soleus muscle by reverse-phase HPLC following extraction in perchloric acid.
4. Determine muscle glycogen content in whole muscle homogenate as glycosyl units after acid hydrolysis by a fluorometric method as previously described¹⁸.

11. Statistics

1. Perform statistical analyses with statistical analyses software.
2. Use a two-way analysis of variance (ANOVA) test to assess statistical differences between values presented in Table 1.
3. Use unpaired Student t tests to assess statistical differences in glucose uptake between EDL and soleus within each group presented in Figure 2. Present data as means ± standard error of the mean (SEM). P < 0.05 is considered statistically significant.

Results

As shown in Figure 2 the basal glucose uptake rates were similar between isolated soleus and EDL muscle from female mice. This has also been reported several times before^12,13,19,20. Glucose uptake increased by ~0.8 and ~0.6 fold reaching 12 and 9 µmol/g protein/h in soleus and EDL muscle, respectively, in response to a submaxi...

Discussion

Intact regulation of glucose uptake in skeletal muscle is important for preserving overall health¹. Thus, investigation of muscle glucose uptake often serves as a primary readout when evaluating various health-altering interventions. Here we describe an ex vivo method for measuring glucose uptake in isolated and incubated soleus and EDL muscle from mice in response to insulin and electrically-induced contractions. The method is quick and reliable and allows a precise control of the surrounding mil...

Materials

Name	Company	Catalog Number	Comments
[14C]D-mannitol	American Radiolabeled Chemicals, Inc.	ARC 0127
[3H]2-deoxy-D-glucose	American Radiolabeled Chemicals, Inc.	ART 0103A
2-Deoxy-D-glucose	Sigma	D8375
4-0 USP non-sterile surgical nylon suture	Harvard Apparatus	51-7698
Streptavidin/HRP (Conjugate)	DAKO	P0397	Used to detect ACC protein
Akt2 antibody	Cell Signaling	3063
AMPKα2 antibody	Santa Cruz	SC-19131
aprotinin	Sigma	A1153
benzamidine	Sigma	B6505
Bovine serum albumin (BSA)	Sigma	A7030
CaCl2	Merck	1020831000
Calibration kit (force)	Danish Myo Technology A/S	300041
Chemiluminescence	Millipore	WBLUF0500
D-Glucose	Merck	1084180100
D-Mannitol	Sigma	M4125
Data collection program	National Instruments	LabVIEW software version 7.1
Dialysis tubing	Visking	DTV.12000.09 Size No.9
Digital imaging system	BioRad	ChemiDoc MP
EDTA	Sigma EDS	E9884
EGTA	Sigma	E4378
Electrical Pulse Stimulator	Digitimer	D330 MultiStim System
Glycerol	Sigma	G7757
HEPES	Sigma	H7637
IGEPAL CA-630	Sigma	I8896
Insulin	Novo Nordisk	Actrapid, 100 IE/mL
KCl	Merck	1049361000
KH2PO4	Merck	104873025
leupeptin	Sigma	L2884
MgSO4	Merck	1058860500
Muscle Strip Myograph System	Danish Myo Technology A/S	Model 820MS
Na-Orthovanadate	Sigma	S6508
Na-Pyrophosphate	Sigma	221368
Na-Pyruvate	Sigma	P2256
NaCl	Merck	106041000
NaF	Sigma	S1504
NaHCO3	VWR	27778260
pACC Ser212 antibody	Cell Signaling	3661
pAkt Thr308 antibody	Cell Signaling	9275
pAMPK Thr172 antibody	Cell Signaling	2531
phenylmethylsulfonylfluoride	Sigma	P7626
Platinum electrodes	Danish Myo Technology A/S	300145
pTBC1D4 Ser588 antibody	Cell Signaling	8730
Scintillation counter	Perkin Elmer	Tri-Carb-2910TR
Scintillation fluid	Perkin Elmer	6013329
Statistical analyses software	Systat	SigmaPlot version 14
TBC1D4 antibody	Abcam	ab189890
TissueLyser II	Qiagen	85300
Ultrapure water	Merck	Milli-Q Reference A+ System
β-glycerophosphate	Sigma	G9422