Name: measurement of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle from mice
Uploaded: 2021-05-16T13:00:00
Description: Watch this Scientific Journal Video about Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice at JoVE.com

This work was supported by grants from the Danish Council for Independent Research - Medical Sciences (FSS8020-00288B) and the Novo Nordisk Foundation (NNF160C0023046). This work was also supported by a research grant to Rasmus Kj&#248;bsted from the Danish Diabetes Academy, which is funded by the Novo Nordisk Foundation, grant number NNF17SA0031406. The authors would like to thank Karina Olsen, Betina Bolmgren, and Irene Bech Nielsen (Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen) for their skilled technical assistance.

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.&#160;
1. Preparation of the experimental apparatus and suture loops
NOTE: For this study, use an integrated muscl...

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...

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 performance1,2, 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 vivo3,4. 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&#160;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.&#160;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 analogues8,9,10.
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 analogues11,12,13. 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.&#160;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 sensitivity12,14. 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.&#160;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 muscle16,17.
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&#160;muscle. The results indicate that glucose supplementation to the incubation buffer is essential to preserve metabolic viability of muscle incubated for 1 hour.

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			<td>[14C]D-mannitol</td>
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			<td>[3H]2-deoxy-D-glucose&#160;</td>
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			<td>4-0 USP non-sterile surgical nylon suture</td>
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			<td>51-7698</td>
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			<td>Streptavidin/HRP (Conjugate)</td>
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			<td>AMPK&#945;2 antibody</td>
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			<td>SC-19131</td>
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			<td>aprotinin</td>
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			<td>A1153</td>
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			<td>benzamidine</td>
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			<td>B6505</td>
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			<td>Bovine serum albumin (BSA)</td>
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			<td>A7030</td>
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			<td>CaCl2</td>
			<td>Merck</td>
			<td>1020831000</td>
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			<td>Calibration kit (force)</td>
			<td>Danish Myo Technology A/S</td>
			<td>300041</td>
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			<td>Chemiluminescence</td>
			<td>Millipore</td>
			<td>WBLUF0500</td>
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			<td>D-Glucose</td>
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			<td>1084180100</td>
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			<td>D-Mannitol</td>
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			<td>M4125</td>
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			<td>Data collection program</td>
			<td>National Instruments</td>
			<td>LabVIEW software version 7.1</td>
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			<td>Dialysis tubing</td>
			<td>Visking</td>
			<td>DTV.12000.09 Size No.9</td>
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			<td>Digital imaging system</td>
			<td>BioRad</td>
			<td>ChemiDoc MP</td>
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			<td>EDTA</td>
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			<td>E9884</td>
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			<td>EGTA</td>
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			<td>E4378</td>
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			<td>Electrical Pulse Stimulator</td>
			<td>Digitimer</td>
			<td>D330 MultiStim System</td>
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			<td>Glycerol</td>
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			<td>G7757</td>
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			<td>HEPES</td>
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			<td>H7637</td>
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			<td>IGEPAL CA-630&#160;</td>
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			<td>I8896</td>
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			<td>Insulin</td>
			<td>Novo Nordisk</td>
			<td>Actrapid, 100 IE/mL</td>
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			<td>KCl</td>
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			<td>Merck</td>
			<td>104873025</td>
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			<td>leupeptin</td>
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			<td>L2884</td>
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			<td>MgSO4</td>
			<td>Merck</td>
			<td>1058860500</td>
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			<td>Muscle Strip Myograph System</td>
			<td>Danish Myo Technology A/S</td>
			<td>Model 820MS</td>
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			<td>Na-Orthovanadate</td>
			<td>Sigma</td>
			<td>S6508</td>
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			<td>Na-Pyrophosphate</td>
			<td>Sigma</td>
			<td>221368</td>
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			<td>Na-Pyruvate</td>
			<td>Sigma</td>
			<td>P2256</td>
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			<td>NaCl</td>
			<td>Merck</td>
			<td>106041000</td>
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			<td>NaF</td>
			<td>Sigma</td>
			<td>S1504</td>
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			<td>NaHCO3</td>
			<td>VWR</td>
			<td>27778260</td>
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			<td>pACC Ser212 antibody</td>
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			<td>3661</td>
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			<td>pAkt Thr308 antibody</td>
			<td>Cell Signaling</td>
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			<td>pAMPK Thr172 antibody</td>
			<td>Cell Signaling</td>
			<td>2531</td>
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			<td>phenylmethylsulfonylfluoride</td>
			<td>Sigma</td>
			<td>P7626</td>
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			<td>Platinum electrodes</td>
			<td>Danish Myo Technology A/S</td>
			<td>300145</td>
			<td></td>
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			<td>pTBC1D4 Ser588 antibody</td>
			<td>Cell Signaling</td>
			<td>8730</td>
			<td></td>
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			<td>Scintillation counter</td>
			<td>Perkin Elmer</td>
			<td>Tri-Carb-2910TR</td>
			<td></td>
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			<td>Scintillation fluid&#160;</td>
			<td>Perkin Elmer</td>
			<td>6013329</td>
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			<td>Statistical analyses software</td>
			<td>Systat</td>
			<td>SigmaPlot version 14</td>
			<td></td>
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			<td>TBC1D4 antibody</td>
			<td>Abcam</td>
			<td>ab189890</td>
			<td></td>
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		<tr>
			<td>TissueLyser II&#160;</td>
			<td>Qiagen</td>
			<td>85300</td>
			<td></td>
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			<td>Ultrapure water</td>
			<td>Merck</td>
			<td>Milli-Q Reference A+ System</td>
			<td></td>
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		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma</td>
			<td>G9422</td>
			<td></td>
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measurement of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle from mice

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&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

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 before12,13,19,20. Glucose uptake increased by ~0.8 and ~0.6 fold reaching 12 and 9 &#181;mol/g protein/h in soleus and EDL muscle, respectively, in response to a submaxi...

Watch this Scientific Journal Video about Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice at JoVE.com

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

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.

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 ...

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