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.

The authors have nothing to disclose

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 milieu of the incubated muscle that permits accurate investigations of muscle glucose uptake rates isolated from the potentially confounding influence of hormones and substrates that can be found in the blood. The method has been used for several years in many studies and is widely adopted by the muscle research community.
The ex vivo incubation model has generally been considered a method to assess glucose transport capacity rather than glucose uptake in skeletal muscle. Glucose transport capacity in incubated muscle can be determined by measuring accumulated D-glucose over a period of time. However, this poses a problem as D-glucose is quickly metabolized in the muscle cell following uptake. To circumvent this problem, the glucose analogue 3-O-Methyl-D-glucose (3-MG) has been widely used to assess glucose transport capacity, as 3-MG is not further metabolized inside the cell after being transported across the cell surface membrane. Thus, the initial rate of intracellular accumulated 3-MG serves as an index of cellular glucose transport capacity per se because it is not affected by other steps in the glucose metabolic pathways. However, the use of 3-MG may constitute a problem as 3-MG will accumulate thereby reducing the transmembrane gradient for 3-MG and subsequently reduce further uptake. Thus, to obtain a measure of the membrane transport capacity, the initial rate of 3-MG uptake must be estimated. In particular when transport capacity is high this may pose a problem due to&#160;efflux of 3-MG from the muscle29,30. The potential problem with 3-MG efflux can be avoided using 2-deoxy-D-glucose (2-DG). Following transport into skeletal muscle, 2-DG is phosphorylated by hexokinase II to 2-deoxy-D-glucose-6-phosphate (2-DG-6P). As skeletal muscle lacks glucose-6-phophatase and GLUT4 cannot transport phosphorylated 2-DG, 2-DG-6P will be trapped within the muscle cell. In contrast to glucose-6-phosphate, 2-DG-6P is a very weak allosteric inhibitor of hexokinase II30 which helps to maintain the transmembrane gradient for 2-DG. Thus, observations have shown that 2-DG uptake in incubated (rat) muscle remains linear until the intracellular 2-DG-6P concentration exceeds 30 mM, a concentration that lowers hexokinase II activity30. Moreover, in incubated mouse skeletal muscle 2-DG uptake remains linear for ~30 min when temperatures of the incubation buffers are 37&#176;C or less31. This suggests that 2-DG can be used to measure glucose transport capacity rather than glucose uptake in incubated muscle except for situations where 2-DG-6P concentrations become very high (e.g. observed during incubations &#62;2 hours with 1 mM 2-DG and a maximal insulin concentration29,30). The idea that 2-DG uptake likely reflects glucose transport capacity is also supported by findings showing that maximal insulin-stimulated 2-DG uptake is similar in incubated muscle from wild-type mice and mice that overexpress hexokinase II32. A potential concern when using 2-DG for muscle glucose transport measurements during contraction is an increase in the intracellular glucose-6-phosphate concentration due to an elevated rate of glycogenolysis. However, since a linear increase in (rat) muscle 2-DG uptake is observed during contraction over time29, this indicates that the accumulation of glucose-6-phosphate from the breakdown of glycogen during contraction does not interfere with hexokinase II activity and thus 2-DG uptake rates. Based on this, 2-DG seems well suited for measurements of glucose transport in isolated skeletal muscle during insulin and contraction considering the limitations of 3-MG.
Although it is generally assumed that 2-DG is not further metabolized following phosphorylation by hexokinase II, it has been reported that some 2-DG is directed towards and incorporated into muscle glycogen. Thus, during a 2 h normoglycemic hyperinsulinemic clamp in rats ~30% of the 2-DG taken up by skeletal muscle is incorporated into glycogen33. Therefore, it could be reasoned that rates of insulin-stimulated 2-DG uptake in incubated skeletal muscle are underestimated if accumulation of 2-DG in glycogen is neglected. We have determined that the herein described protocol on how to prepare muscle lysates (supernatant) for subsequent analyses of 2-DG uptake rates in prior incubated skeletal muscle is not affected by the potential incorporation of 2-DG into glycogen. It could be argued that glycogen accumulates in the pellet when centrifuging whole muscle homogenate to generate lysate for 2-DG uptake measurements. However, when comparing the levels of radioactivity in insulin-stimulated whole muscle homogenate vs. lysate we do not detect any significant difference (unpublished data). This suggests that incubating mouse muscle for 10 min in 1 mM of 2-DG does not cause a detectable accumulation of 2-DG in glycogen.
Determination of skeletal muscle 2-DG uptake without considering that 2-DG is distributed in both the extra- and intracellular space will lead to an overestimation of 2-DG uptake. To circumvent this, L-glucose should be used as an extracellular marker as this is not transported across the cell membrane but otherwise exhibits similar properties as D-glucose including mass, solubility, passive diffusion, binding, etc. Due to the excessive costs of manufacturing and thus purchasing L-glucose, mannitol is typically used as an extracellular marker since mannitol is not taken up by the muscle cell, is relatively inexpensive and is estimated to have a somewhat similar extracellular distribution volume as glucose and 2-DG34.
Intrinsic cellular and molecular clocks seem to play an essential role for the regulation of whole-body metabolism and energy homeostasis35. It has been observed that muscle-specific knockout of the core clock gene Bmal1 impairs insulin-stimulated glucose uptake in isolated mouse skeletal muscle36. Furthermore, submaximal insulin-stimulated glucose uptake of isolated skeletal muscle exhibits circadian rhythm with the lowest and highest insulin response in the middle of the light and dark phase, respectively37. Based on these findings, it is therefore important to incorporate time of animal sacrifice into the experimental design to increase the reproducibility of data.
A major disadvantage of the ex vivo method is the lack of capillary flow in the isolated muscle. This means that delivery and removal of various substrates fully depend on simple diffusion between the muscle fibers and the surrounding environment. Consequently, validation of the metabolic viability of isolated muscle incubated ex vivo has been focused on diffusion limitations of oxygen to superficial as well as deep muscle fibers. Thus, incubated muscle, particularly highly metabolic mouse muscle, tends to develop hypoxic cores in which breakdown of glycogen occurs16,17,38. In a detailed review by Bonen and colleagues15, it was suggested that hypoxic cores of incubated muscle likely develop when incubating muscle too thick to be properly oxygenated especially when incubating at temperatures of &#8805; 37 &#176;C. This led to the recommendation that only thin and cylindrical mouse muscles, such as soleus and EDL, should be used for incubations at 25-30 &#176;C to avoid development of hypoxic cores. This indicates that thickness and geometry rather than mass are important factors to consider when incubating mouse skeletal muscles. In addition, it was recommended that incubation temperature, muscle thickness as well as ATP, phosphocreatine, and glycogen content and/or release of lactate should be measured and reported routinely to evaluate the viability of the incubated muscle15. To our knowledge, such complete analyses of incubated muscle have mainly been reported for rat muscle39,40,41,42 and only to a limited extent reported for mouse muscle16,17. To increase insight into various metabolic markers of viability in incubated mouse skeletal muscle, we assessed possible changes in intracellular content of lactate, creatine, phosphocreatine, adenosine nucleotides, glycogen, and AMPK signaling in soleus and EDL muscle following 1 hour of incubation in glucose- or pyruvate-supplemented KRH buffer. Similar to previous findings in incubated mouse soleus and EDL muscle17, we observed that glycogen levels decreased when muscles were incubated in glucose-free media. This indicates that the absence of glucose during incubation promotes glycogen breakdown and the subsequent entry of glucose-6-phosphate into glycolysis that may act to secure ATP production in particular if oxygen supply is inadequate. Furthermore, we found an overall reduction in the ATP and ADP nucleotide pools in incubated soleus and EDL muscle. This may imply that oxygen supply is not entirely sufficient to meet the demand of incubated mouse muscle both in the presence and absence of glucose. Since the oxidative soleus muscle relies more on oxygen for ATP production compared to the glycolytic EDL muscle, this would suggest that the soleus muscle is affected to a larger extent by the incubation procedure. In agreement, we found that the intracellular levels of IMP as well as AMPK signaling were markedly&#160;increased in soleus compared to EDL muscle when incubated in the absence of glucose. This signifies that the soleus muscle exhibits&#160;a higher degree of metabolic stress during incubation, which must be taken into consideration when evaluating data from the ex vivo mouse muscle model.
Cultured muscle cells including immortalized L6 and C2C12 as well as primary human myotubes are commonly used as a surrogate for mature skeletal muscle to study the effects of various genetic and pharmacological manipulations on insulin-stimulated muscle glucose uptake. However, in several ways cultured muscle cells do not resemble mature skeletal muscle and when comparing the two model systems numerous differences become apparent. These include differences in protein expression, dimensional structure, surrounding environment, proliferation and differentiation state, fiber type composition, metabolic processes and functional properties43 all of which may affect how the muscle cell regulates glucose uptake in response to various stimuli. Typically, greater relative effects of insulin on glucose uptake rates are observed in mature skeletal muscle compared to cultured muscle cells44 which may indicate that cultured muscle cells to some extent lack the machinery responsible for regulating glucose uptake rates, including a high level of expression of the insulin-sensitive glucose transporter GLUT443. Considering the limitations and caveats associated with the use of either cultured muscle cells and&#160;isolated skeletal muscle, it is therefore likely advantageous to take on a combined approach when studying various muscle metabolic processes such as glucose uptake.
Soleus and EDL muscles are typically regarded as representative of slow- and fast-twitch muscles, respectively. Therefore, these muscle types are ideal for mechanical studies seeking to investigate interventions that affect muscle force and fatigue development. Moreover, soleus muscle is typically reported to have enhanced insulin sensitivity and responsiveness compared to EDL muscle and thus, interventions that target muscle insulin sensitivity may affect soleus and EDL differently. In addition, the relative distribution of the different AMPK complexes differs between the soleus and EDL muscle that seems to affect the ability of AMPK activating compounds to increase muscle glucose uptake. Thus, the greatest insight into the regulation of muscle glucose uptake is likely achieved if both the soleus and EDL muscles are used during experimentation with the ex vivo incubation model.
The herein described method relates glucose uptake to the amount of total protein abundance determined in each muscle sample after homogenization. It is our experience that variation decreases when relating glucose uptake per amount of muscle protein instead of muscle weight (unpublished data). Furthermore, homogenization of muscle samples in a buffer used for various biochemical assays makes it possible to determine both glucose uptake, myocellular signaling and enzyme&#160;activities in the same sample preparation45,46. This will often decrease the amount of mice used for a study. Nevertheless, glucose uptake can easily be determined in skeletal muscle samples that are dissolved by heating in sodium hydroxide (NaOH) followed by neutralization with hydrogen chloride20. Since treatment of muscle with NaOH interferes with measurements of total muscle protein concentration, glucose transport measured by this procedure must be related to muscle weight.
Based on various optimizations, our glucose uptake incubation buffer contains a specific activity of 0.028 MBq/mL of [3H]2-deoxy-D-glucose and 0.0083 MBq/mL of [14C]Mannitol. On one hand, this lowers the amount of lysate (150 out of 400 &#181;L) needed in order to get sufficient and reliable radioactivity measurements. On the other hand, it increases the amount of radioactive [3H]2-deoxy-D-glucose and [14C]mannitol needed for each experiment, which increases experimental costs. Thus, for any experimental setup it is possible to regulate the amount of radioactivity used in the glucose uptake incubation media to fit specific requirements. However, care must be taken not to decrease amount of radioactivity in the incubation buffer to an extent that makes radioactivity measurements unreliable. This is ensured by keeping radioactivity in samples higher than the specified detection limits of the liquid scintillation counting machinery used.

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.

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

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 submaximally effective insulin concentration (100 &#181;U/mL). This increase was even higher (~4 and ~2 fold reaching 33 and 19 &#181;mol/g protein/h in soleus and EDL muscle, respectively) when muscles were stimulated with a maximally effective insulin concentration (10 mU/mL). Moreover, both submaximal and maximal insulin-stimulated glucose uptake were significantly higher in soleus muscle indicating that soleus muscle exhibits enhanced insulin sensitivity and responsiveness compared to EDL muscle. This may be related to the higher expression of the glucose transporter 4 (GLUT4) as well as the insulin signaling transducer protein kinase B (Akt) in soleus muscle compared to EDL muscle10,21,22,23,24.
Contraction-induced glucose uptake was significantly higher in EDL muscle compared to soleus muscle (Figure 2) as also previously reported13,19. Thus, glucose uptake increased by ~2 and ~2.5 fold reaching 14 and 22 &#181;mol/g protein/h in soleus and EDL muscle, respectively, in response to electrically-induced contractions. Figure 3 shows maximal muscle force production in soleus and EDL muscle during the 10 min stimulation period. As seen and previously reported19, the EDL muscle generates more force (225 mN in EDL vs. 150 mN in soleus) during the initial part of the stimulation period. On the other hand, the EDL muscle exhibits a faster decline in force production compared to the soleus muscle later on in the stimulation period. These findings are likely due to the difference in fiber type distribution between the soleus (type 1 &#62; type 2) and EDL (type 2 &#62; type 1) muscle25 as type 2 fibers generate more force but fatigue faster compared to type 1 fibers26,27.
To evaluate the effect of insulin and contraction on intracellular signaling in isolated soleus and EDL muscle, phosphorylation of Akt Thr308, TBC1 domain family member 4 (TBC1D4) Ser588, AMPK&#945; Thr172 and acetyl-CoA carboxylase (ACC) Ser212 was performed by the western blotting technique&#160;(Figure 4). As expected, the submaximally and maximally effective insulin concentration induced an increase in the phosphorylation of Akt Thr308 and TBC1D4 Ser588 while contraction induced an increase in the phosphorylation of AMPK&#945; Thr172 and ACC Ser212. Neither insulin nor contraction led to a change in the total protein content of Akt2, TBC1D4, AMPK&#945;2 and ACC in soleus and EDL muscle (Figure 4).
Examining various markers of metabolic viability of incubated soleus and EDL muscle, we observed an overall reduction in the levels of adenosine nucleotides (ATP, ADP, AMP) (~15-25%) as well as creatine (~10-35%) regardless of whether the muscles were incubated in the presence of pyruvate or glucose compared to non-incubated muscles (Table 1). On the other hand, the drop in glycogen levels observed in soleus and EDL muscles incubated with pyruvate was prevented if the muscles were incubated with glucose. Interestingly though, we observed that the inosine monophosphate (IMP) levels increased several fold&#160;but only in incubated soleus muscle. IMP levels typically increase in muscle during severe metabolic stress as the muscle attempts to prevent AMP accumulation by converting AMP into IMP in order to maintain the ATP/ADP ratio28. This indicates that the soleus muscle is somewhat more metabolically stressed compared to the EDL muscle during incubation. This notion is also supported by findings of elevated AMPK&#945; Thr172 and ACC Ser212 phosphorylation in the soleus muscle incubated with pyruvate (Figure 5). Importantly, the observed increase in IMP levels as well as AMPK&#945; Thr172 and ACC Ser212 phosphorylation decrease when the soleus muscle is incubated with glucose. Hence, it seems advantageous to incubate isolated skeletal muscle in a glucose-containing buffer to minimize fluctuations in adenosine nucleotides and prevent a drop in muscle glycogen&#160;when muscles are incubated for a prolonged period of time. With regards to 2-deoxyglucose uptake, we have evidence to suggest that incubating muscles for 6 to 8 h in the presence of pyruvate will increase basal/resting glucose uptake rates. Incubating muscles in a media containing glucose seems to prevent such increase in glucose uptake (unpublished data).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61398/61398fig01.jpg" /> 
Figure 1: Incubation system. (A) Myograph system with four single incubation chambers. (B) Customized incubation hooks. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61398/61398fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61398/61398fig02.jpg" /> 
Figure 2: Glucose uptake in isolated mature skeletal muscle from mice. 2-deoxyglucose uptake was determined in isolated soleus (black bars) and EDL (gray bars) muscles in response to a submaximally effective insulin concentration (100 &#181;U/mL), a maximally effective insulin concentration (10 mU/mL), and electrically-induced contractions (0.2 ms pulse, 100 Hz, 1 s/15s, 30 V, 10 min). Data were analyzed by Students t test within each group. ###p&#60;0.001, ##p&#60;0.01 vs. soleus muscle. Values are means &#177; SEM. n = 4-6 per group. h, hour. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61398/61398fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61398/61398fig03.jpg" /> 
Figure 3: Muscle force curves in response to electrically induced contractions. Peak force production during electrical stimulation was calculated for the soleus (black dots) and EDL (gray dots) muscle. Each single value corresponds to an average of the last 500 ms of each 1-s stimulation period. Values are means &#177; SEM. n = 5-6 per group. s, second. mN, milli-Newton. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61398/61398fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61398/61398fig04.jpg" /> 
Figure 4: Representative western blots of Akt Thr308, TBC1D4 Ser588, AMPK&#945; Thr172 and ACC Ser212 phosphorylation as well as Akt2, TBC1D4, AMPK&#945;2 and ACC protein. Western blot analyses were performed on the mouse soleus and EDL muscle samples described in Figure 2. B, basal. S, submaximally effective insulin. M, maximally effective insulin. C, contraction. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61398/61398fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61398/61398fig05.jpg" /> 
Figure 5: Representative western blots of AMPK&#945; Thr172 and ACC Ser212 phosphorylation as well as AMPK&#945;2 and ACC protein. Western blot analyses were performed on the mouse soleus and EDL muscle samples described in Table 1. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61398/61398fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:font-size="8px" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">non-incubated</td>
			<td colspan="2">Incubated 1h with pyruvate</td>
			<td colspan="2">Incubated 1h with glucose</td>
			<td rowspan="2">Main effects</td>
			<td rowspan="2">Interaction</td>
		</tr>
		<tr>
			<td></td>
			<td>Soleus</td>
			<td>EDL</td>
			<td>Soleus</td>
			<td>EDL</td>
			<td>Soleus</td>
			<td>EDL</td>
		</tr>
		<tr>
			<td>Lactate</td>
			<td>1.00 &#177; 0.01</td>
			<td>1.00 &#177; 0.02</td>
			<td>0.96 &#177; 0.03</td>
			<td>0.98 &#177; 0.02</td>
			<td>1.05 &#177; 0.01</td>
			<td>0.99 &#177; 0.01</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cr</td>
			<td>1.00 &#177; 0.04</td>
			<td>1.00 &#177; 0.06</td>
			<td>0.64 &#177; 0.07 ###</td>
			<td>0.76 &#177; 0.05 ###</td>
			<td>0.76 &#177; 0.07 ##</td>
			<td>0.88 &#177; 0.09 ##</td>
			<td>p &#60; 0.001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PCr</td>
			<td>1.00 &#177; 0.19</td>
			<td>1.00 &#177; 0.06</td>
			<td>0.80 &#177; 0.12</td>
			<td>1.13 &#177; 0.14</td>
			<td>0.65 &#177; 0.31</td>
			<td>0.75 &#177; 0.08</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PCr/(Cr + PCr)</td>
			<td>1.00 &#177; 0.20</td>
			<td>1.00 &#177; 0.07</td>
			<td>1.17 &#177; 0.11</td>
			<td>1.25 &#177; 0.12</td>
			<td>0.78 &#177; 0.36</td>
			<td>0.92 &#177; 0.11</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>1.00 &#177; 0.03</td>
			<td>1.00 &#177; 0.02</td>
			<td>0.72 &#177; 0.03 ###,&#167;&#167;&#167;</td>
			<td>0.99 &#177; 0.03 *</td>
			<td>0.81 &#177; 0.06 ###</td>
			<td>0.85 &#177; 0.04 ##</td>
			<td>-</td>
			<td>p &#60; 0.001</td>
		</tr>
		<tr>
			<td>ADP</td>
			<td>1.00 &#177; 0.04</td>
			<td>1.00 &#177; 0.04</td>
			<td>0.75 &#177; 0.05 ###</td>
			<td>0.92 &#177; 0.03 ###</td>
			<td>0.84 &#177; 0.04 ##</td>
			<td>0.86 &#177; 0.03 ##</td>
			<td>p &#60; 0.001</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AMP</td>
			<td>1.00 &#177; 0.11</td>
			<td>1.00 &#177; 0.12</td>
			<td>0.85 &#177; 0.15</td>
			<td>0.79 &#177; 0.13</td>
			<td>0.84 &#177; 0.18</td>
			<td>0.75 &#177; 0.13</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>IMP</td>
			<td>1.00 &#177; 0.17</td>
			<td>1.00 &#177; 0.30</td>
			<td>4.43 &#177; 0.67 ###,&#167;&#167;&#167;</td>
			<td>0.72 &#177; 0.29</td>
			<td>3.33 &#177; 1.25 ##,&#167;</td>
			<td>1.08 &#177; 0.01</td>
			<td>-</td>
			<td>p &#60; 0.001</td>
		</tr>
		<tr>
			<td>AMP/ATP ratio</td>
			<td>1.00 &#177; 0.12</td>
			<td>1.00 &#177; 0.13</td>
			<td>1.18 &#177; 0.22</td>
			<td>0.81 &#177; 0.16</td>
			<td>1.06 &#177; 0.23</td>
			<td>0.90 &#177; 0.19</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>1.00 &#177; 0.08</td>
			<td>1.00 &#177; 0.12</td>
			<td>0.74 &#177; 0.07 (#),*</td>
			<td>0.80 &#177; 0.12 (#),*</td>
			<td>1.12 &#177; 0.10</td>
			<td>1.10 &#177; 0.03</td>
			<td>p = 0.035</td>
			<td>-</td>
		</tr>
		<tr>
			<td>pAMPK Thr172 / AMPK&#945;2</td>
			<td>1.00 &#177; 0.10</td>
			<td>1.00 &#177; 0.12</td>
			<td>3.26 &#177; 0.58 ###,***,&#167;&#167;&#167;</td>
			<td>1.55 &#177; 0.27</td>
			<td>1.68 &#177; 0.19 #</td>
			<td>1.36 &#177; 0.19</td>
			<td>-</td>
			<td>p = 0.002</td>
		</tr>
		<tr>
			<td>pACC Ser212 / ACC</td>
			<td>1.00 &#177; 0.18</td>
			<td>1.00 &#177; 0.12</td>
			<td>2.22 &#177; 0.58 ###,**,&#167;&#167;</td>
			<td>0.96 &#177; 0.21</td>
			<td>0.99 &#177; 0.15</td>
			<td>0.83 &#177; 0.09</td>
			<td>-</td>
			<td>p = 0.030</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of metabolic viability of mouse soleus and EDL muscles incubated for 1 hour in the presence of 2 mM pyruvate or 5 mM glucose. Non-incubated muscles were dissected from anaesthetized and fed animals before being frozen in liquid nitrogen. Separate muscles were incubated for 1 h in KRH buffer supplemented with BSA (0.1%), Na-pyruvate (2 mM) and D-mannitol (8 mM) while others were incubated for 1 h in KRH buffer supplemented with BSA (0.1%), D-glucose (5 mM) and D-mannitol (5 mM) before frozen in liquid nitrogen. Data were converted into relative units to highlight observed changes in various markers of metabolic viability. Absolute values from non-incubated mouse soleus and EDL muscles are given below. Lactate (mmol/kg w.w): 137.53soleus; 139.05EDL. Cr (mmol/kg w.w): 9.35soleus; 8.98EDL. PCr (mmol/kg w.w): 1.50soleus; 5.20EDL. PCr/(PCr+Cr): 13.98soleus; 37.30EDL. ATP (mmol/kg w.w): 3.07soleus; 4.24EDL. ADP (mmol/kg w.w): 0.60soleus; 0.51EDL. AMP (mmol/kg w.w): 0.18soleus; 0.08EDL. IMP (mmol/kg w.w): 0.07soleus; 0.14EDL. AMP/ATP ratio: 0.06soleus; 0.02EDL. Glycogen (pmol/&#181;g protein): 77.01soleus; 67.56EDL. Data were analyzed by a two-way ANOVA within each group. ###p&#60;0.001, ##p&#60;0.01, and #p&#60;0.05 vs. non-incubated. ***p&#60;0.001, **p&#60;0.01, and *p&#60;0.05 vs incubated 1 h with glucose. &#167;&#167;&#167;p&#60;0.001, &#167;&#167;p&#60;0.01, and &#167;p&#60;0.05 vs EDL. Values are means &#177; SEM. n = 12 in non-incubated group, n = 4-6 in incubated groups. Cr, Creatine; PCr, Phosphocreatine; w.w, wet weight; h, hour.

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 ...

measurement-insulin-contraction-stimulated-glucose-uptake-isolated

Universidad San Sebastian

Research

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Biology

5.7K Views.  University of Copenhagen. Het ex vivo model is een nuttige methode voor het evalueren van de impact van verschillende fysiologische interventies en farmacologische verbindingen en spierglucose opname. Deze techniek maakt de nauwkeurige beoordeling van glucoseopname in geïsoleerde skeletspieren van muizen mogelijk in afwezigheid van verstorende factoren die een intact diermodel kunnen verstoren. Schakel voordat u aan een experiment begint het geïntegreerde spierstripsysteem in en verwarm de kamers tot 30 graden Celsi...

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

Hyperinsulinemic-euglycemic Clamps in Conscious, Unrestrained Mice

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold standard" method for assessing insulin action in vivo. During an insulin clamp, hyperinsulinemia is achieved by a constant insulin infusion. Euglycemia is maintained via a concomitant glucose infusion at a variable rate. This variable glucose infusion rate (GIR) is determined by measuring blood glucose at brief intervals throughout the experiment and adjusting the GIR accordingly. The GIR is indicative of whole-body insulin action, as mice with enhanced insulin action require a greater GIR. The insulin clamp can incorporate administration of isotopic 2[14C]deoxyglucose to assess tissue-specific glucose uptake and [3-3H]glucose to assess the ability of insulin to suppress the rate of endogenous glucose appearance (endoRa), a marker of hepatic glucose production, and to stimulate the rate of whole-body glucose disappearance (Rd).
The miniaturization of the insulin clamp for use in genetic mouse models of metabolic disease has led to significant advances in diabetes research. Methods for performing insulin clamps vary between laboratories. It is important to note that the manner in which an insulin clamp is performed can significantly affect the results obtained. We have published a comprehensive assessment of different approaches to performing insulin clamps in conscious mice1 as well as an evaluation of the metabolic response of four commonly used inbred mouse strains using various clamp techniques2. Here we present a protocol for performing insulin clamps on conscious, unrestrained mice developed by the Vanderbilt Mouse Metabolic Phenotyping Center (MMPC; URL: www.mc.vanderbilt.edu/mmpc). This includes a description of the method for implanting catheters used during the insulin clamp. The protocol employed by the Vanderbilt MMPC utilizes a unique two-catheter system3. One catheter is inserted into the jugular vein for infusions. A second catheter is inserted into the carotid artery, which allows for blood sampling without the need to restrain or handle the mouse. This technique provides a significant advantage to the most common method for obtaining blood samples during insulin clamps which is to sample from the severed tip of the tail. Unlike this latter method, sampling from an arterial catheter is not stressful to the mouse1. We also describe methods for using isotopic tracer infusions to assess tissue-specific insulin action. We also provide guidelines for the appropriate presentation of results obtained from insulin clamps.

The hyperinsulinemic-euglycemic clamp, or insulin clamp, is the gold standard for assessing insulin action in vivo. A method for performing insulin clamps in mice is described. This includes a method for arterial catheterization that permits experiments to be performed in conscious, unrestrained mice with minimal stress.

Type 2 diabetes is characterized by a defect in insulin action. The hyperinsulinemic-euglycemic clamp, or insulin clamp, is widely considered the "gold ...

Medicine

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic factors affecting glucose uptake. The measurement of intracellular fluorescence is a widely used method to test the uptake of fluorescently-labeled glucose (FD-glucose) in cells in vitro, or for imaging glucose-consuming tissues in vivo. This assay assesses glucose uptake at a chosen time point. The intracellular analysis assumes that the metabolism of FD-glucose is slower than that of endogenous glucose, which participates in catabolic and anabolic reactions and signaling. However, dynamic glucose metabolism also alters uptake mechanisms, which would require kinetic measurements of glucose uptake in response to different factors. This article describes a method for measuring extracellular FD-glucose depletion and validates its correlation with intracellular FD-glucose uptake in cells and tissues ex vivo. Extracellular glucose depletion may be potentially applicable for high-throughput kinetic and dose-dependent studies, as well as identifying compounds with glycemic activity and their tissue-specific effects.

Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues Ex Vivo

Extracellular depletion of fluorescently labeled glucose correlates with glucose uptake and could be used for high-throughput screening of glucose uptake in excised organs and cell cultures.

The ongoing worldwide epidemic of diabetes increases the demand for the identification of environmental, nutritional, endocrine, genetic, and epigenetic ...

Biochemistry

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). Impaired uptake and metabolism of glucose in multiple organ systems due to insulin resistance is a hallmark of type 2 diabetes. The ability of this technique to extract an image-derived input function from the vena cava using an iterative deconvolution method eliminates the requirement of the collection of arterial blood samples. Fitting of tissue and vena cava time activity curves to a two-tissue, three compartment model permits the estimation of kinetic micro-parameters related to the 18F-FDG uptake from the plasma to the intracellular space, the rate of transport from intracellular space to plasma and the rate of 18F-FDG phosphorylation. This methodology allows for multiple measures of glucose uptake and metabolism kinetics in the context of longitudinal studies and also provides insights into the efficacy of therapeutic interventions.

Determining Glucose Metabolism Kinetics Using 18F-FDG Micro-PET/CT

This study describes a protocol that uses 18F-FDG and positron emission tomography/computed tomography (PET/CT) imaging, together with kinetic modelling, to quantify the in vivo, real-time uptake of 18F-FDG into tissues.

This paper describes the use of 18F-FDG and micro-PET/CT imaging to determine in vivo glucose metabolism kinetics in mice (and is transferable to rats). ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Acclimation Prior to an Intraperitoneal Insulin Tolerance Test to Mitigate Stress-Induced Hyperglycemia in Conscious Mice

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that involves measurement of blood glucose levels over time following a single intraperitoneal injection of insulin. Given that it is performed in the conscious state and blood is often collected via a tail snip, it has the potential to elicit a stress response from animals due to anxiety associated with handling and blood collection. As such, a stress-induced rise in blood glucose can occur, making it difficult to detect and interpret the primary endpoint measure, namely an insulin-mediated reduction in blood glucose. This has been seen in many mouse strains, and is quite common in diabetic db/db mice, where glucose levels can increase, rather than decrease, after insulin administration. Here, we describe a method of acclimating mice to handling, injections and blood sampling prior to performing the insulin tolerance test. We find that this lowers stress-induced hyperglycemia and results in data that more accurately reflects whole body insulin sensitivity.

Stress-induced elevations in glucose levels can confound interpretation of data derived from a conscious intraperitoneal insulin tolerance test in mice. In this article, we describe a method to acclimate the mice to handling, injections and blood sampling prior to performing the insulin tolerance test in order to limit stress-induced hyperglycemia.

The insulin tolerance test is commonly used in metabolic studies to assess whole body insulin sensitivity in rodents. It is a relatively simple test that ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Surgical Installation of Hyperglycemic and Hypoglycemic Clamps in Mice

Hyperglycemic Clamp and Hypoglycemic Clamp in Conscious Mice

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, and adipose tissues (Type2 DM). Insulin injection treats DM patients but leads to hypoglycemia as a side effect. Cortisol and catecholamines are released to activate glucose production from the liver to recover hypoglycemia, called counter-regulatory responses (CRR). In DM research using rodent models, glucose tolerance tests and 2-deoxy-glucose injection are used to measure insulin release and CRR, respectively. However, blood glucose concentrations change persistently during experiments, causing difficulties in assessing net insulin release and CRR. This article describes a method in which blood glucose is kept at 250 mg/dL or 50 mg/dL in conscious mice to compare the release of insulin and CRR hormones, respectively.
Polyethylene tubing is implanted in the mice's carotid artery and jugular vein, and the mice are allowed to recover from the surgery. The jugular vein tubing is connected to a Hamilton syringe with a syringe pump to enable insulin or glucose infusion at a constant and variable rate. The carotid artery tubing is for blood collection. For the hyperglycemic clamp, 30% glucose is infused into the vein, and blood glucose levels are measured from the arterial blood every 5 min or 10 min. The infusion rate of 30% glucose is increased until the blood glucose level becomes 250 mg/dL. Blood is collected to measure insulin concentrations. For hypoglycemic clamp, 10 mU/kg/min insulin is infused together with 30% glucose, whose infusion rate is variable to maintain 50 mg/dL of blood glucose level. Blood is collected to measure counter-regulatory hormones when both glucose infusion and blood glucose reach a steady state. Both hyperglycemic and hypoglycemic clamps have the same surgical procedure and experimental setups. Thus, this method is useful for researchers of systemic glucose metabolism.

Author Spotlight: Investigating the Blood Glucose Homeostasis in Murine Brain Using a Cost-Effective Hyperglycemic And Hypoglycemic Clamp Technique

A hyperglycemic clamp is used for measuring insulin release with a maintained higher blood glucose concentration. A hypoglycemic clamp is for measuring glucose production induced by counter-regulatory responses. Both methods use the same surgical procedure. Here, we present a clamp technique to assess systemic glucose metabolism.

Diabetes mellitus (DM) is caused by insufficient insulin release from the pancreatic &#946;-cells (Type1 DM) and insulin sensitivity in muscles, liver, ...

In Vitro Glucose Depletion Assay: A Fluorescence-Based Method to Quantify Cellular Glucose Uptake via Extracellular Glucose Depletion Measurement

Source: Kumar, S. B. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/63681">Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues&#160;Ex Vivo.</a>&#160;J. Vis. Exp.&#160;(2022)
This video demonstrates an in vitro assay to indirectly measure intracellular glucose uptake. The assay uses a fluorescently-labeled glucose analog to establish a correlation between extracellular glucose depletion and intracellular glucose uptake.

Source: Kumar, S. B. et al., Extracellular Glucose Depletion as an Indirect Measure of Glucose Uptake in Cells and Tissues&#160;Ex Vivo.&#160;J. Vis. ...

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

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