Name: High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds (Video) | JoVE Journal
Uploaded: 2018-01-23T15:00:00.000+00:00
Duration: 12 min 32 s
Description: Learn high-resolution respirometry to measure mitochondrial function in intact pancreatic beta cells using INS-1-derived 832/13 cells. This method evaluates natural compound effects on respiration under insulin stimulatory conditions for diabetes research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The authors would like to thank members of the Tessem and Hancock labs for assistant and scientific discussion. The authors thank Andrew Neilson, PhD (Virginia Tech) for providing the cocoa derived epicatechin monomer fraction. This study was supported by a grant from the Diabetes Action Research and Education Foundation to JST.

1. Cell Culture
<ol>
	<li>Culture INS-1 derived 832/13 beta cells in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 10 mM HEPES, 2 mM Glutamine, 1 mM Sodium Pyruvate, and 0.05 mM 2-mercaptoethanol9,10,11,12,13.</li>
	<li>Remove 832/13 cells from a T75 flask using 2 mL of 0.25% trypsin, incubating at 37 &#176;C for 10 min. Neutralize trypsin by adding 8 mL of complete RPMI 1640 media.</li>
	<li>Count cells using a hemocytometer and dilute 100 &#181;L of the cell volume from step 1.2 in 900 &#181;L of PBS.</li>
	<li>Plate 832/13 cells at a density of 2 x 106 cells/mL in a 6 well dish.</li>
	<li>Culture cells for 48 h in a humidified incubator at 37 &#176;C and 5% CO2 before beginning respiration experiments.</li>
</ol>
2. Preparation of Cells for High-resolution Respirometry
<ol>
	<li>Treating 832/13 cells with cocoa derived epicatechin monomer.
	<ol>
		<li>Culture 832/13 cells for 24 h after plating in a humidified incubator at 37 &#176;C and 5% CO2.</li>
		<li>Change media 24 h after plating, and treat 832/13 cells with vehicle control (3 wells) or 100 nM cocoa monomer (3 wells), followed by culture for an additional 24 h (48 h total) in a humidified incubator at 37 &#176;C and 5% CO2.</li>
		<li>Wash 832/13 cells in 1x Low Glucose Secretion Assay Buffer (SAB) for 5 min. Aspirate buffer, and incubate 832/13 cells in 1x Low Glucose SAB for 3 h, changing 1x Low Glucose SAB every hour.
		<ol>
			<li>Make 10x SAB with 33.32 g of NaCl, 1.73 g of KCl, 0.82 g of KH2PO4, 0.7 g of MgSO4 and add H2O to a final volume of 500 mL. Sterile filter the final solution.</li>
			<li>Make 1x Low Glucose SAB with 10 mL of 10x SAB, 100 &#181;L of 45% glucose, 2 mL of 1 M HEPES, 1 mL of 0.25M CaCl2, 0.57 mL of 35% BSA solution, 0.21 g of NaHCO3 and add H2O to a final volume of 100 mL. Sterile filter the final solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Treating 832/13 cells with curcumin.
	<ol>
		<li>Culture 832/13 cells for 48 h after plating in a humidified incubator at 37 &#176;C and 5% CO2.</li>
		<li>Wash 832/13 cells in 1x Low Glucose SAB for 5 min. Aspirate buffer, and incubate 832/13 cells in 1x Low Glucose SAB for 3 h, changing 1x Low Glucose SAB every hour.</li>
		<li>After 2.5 h of the 3 h incubation in 1x Low Glucose SAB, aspirate buffer and incubate cells in 1x Low Glucose SAB with vehicle control or 40 &#181;M curcumin for 30 min. Following incubation, aspirate the buffer.</li>
	</ol>
	</li>
	<li>Harvesting cells with trypsin for high-resolution respirometry
	<ol>
		<li>After the 3 h incubation in 1x Low Glucose SAB, remove the cells from the plate with 250 &#181;L of 0.25% trypsin per well.</li>
		<li>Combine the cells in trypsin from 3 wells treated with vehicle control or compound of interest with 4 mL of SAB in a 15 mL conical tube.</li>
		<li>Count cells using a hemocytometer and dilute 100 &#181;L of the cell volume from step 2.3.2 in 900 &#181;L of PBS.</li>
		<li>Dilute the appropriate number of cells in 1x Low Glucose SAB to make 3 mL at the appropriate concentration for each chamber. The data demonstrates that a concentration of 1 x 106 cells/mL is the most effective.</li>
	</ol>
	</li>
	<li>Harvesting cells with mechanical dissociation for high-resolution respirometry
	<ol>
		<li>After the 3 h incubation in 1x Low Glucose SAB, add 1 mL of 1x Low Glucose SAB to each well and gently blow cells off the plate with mechanical dissociation by pipetting with a 1000 &#181;L pipette tip.</li>
		<li>Combine the cells from 3 wells treated with vehicle control or compound of interest in a total of 3 mL SAB in a 15 mL conical tube for respiration.</li>
	</ol>
	</li>
</ol>
3. High-resolution Respirometry
<ol>
	<li>Preparation of the high-resolution respirometer.
	<ol>
		<li>Turn on the high-resolution respirometer and connect to the desktop by launching the respirometer analysis program.</li>
		<li>Aspirate 70% ethanol from both chambers. Soak these chambers in 70% ethanol for a minimum of 45 min.</li>
		<li>Wash the chambers twice with 70% ethanol, aspirating after each step.</li>
		<li>Wash the chambers three times with ddH2O, aspirating after each step.</li>
		<li>Rinse the chamber plungers in ddH2O. This cleans off residual ethanol from the plungers and from the titanium port.</li>
	</ol>
	</li>
	<li>Calibration of polarographic oxygen sensors.
	<ol>
		<li>Add 2.4 mL of 1x Low Glucose SAB buffer to each oxygraph chamber, stirring the buffer continuously using magnetic stir bars in the chamber at 750 rpm and 37 &#176;C with a data-recording interval at 2.0 s by pushing the F7 button and opening the tab labeled &#34;Systems&#34;. Push plungers all the way in, and then retract to the wrench aeration setting. Let the machine equilibrate for a minimum of 1 hour until stable oxygen flux is obtained.</li>
		<li>Set the machine at 37 &#176;C for the duration of the experiment. Set polarization voltage to 800 mV, with a gain of 2 by pushing the F7 button and opening the tab labeled &#34;Oxygen, O2&#34;. Equilibrate the oxygen concentration of the SAB buffer for at least 30 min, while the change in oxygen concentration is stable (less than 2 pmol/(s*mL)).</li>
		<li>Following oxygen concentration stabilization, select a region where the change of oxygen concentration is stable to establish background measurement of change in oxygen concentration.
		<ol>
			<li>Select a region by pushing the shift key, left clicking on the mouse and dragging the mouse across the selected region. Click on the letter associated with the selected region and change to &#34;R1&#34; for each trace, corresponding with each of the two chambers.</li>
			<li>Double click on the &#34;O2 Calibration&#34; box in the bottom left and right corners of the screen, click the &#34;select mark&#34; button for the &#34;air calibration&#34; as R1 and then select &#34;calibrate and copy to clipboard&#34; for both chambers.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Evaluation of respiration of 832/13 beta cells prepared in steps 2.1.5 or 2.2.5.
	<ol>
		<li>Load 2.4 mL of sample in each chamber (one chamber with control vehicle treated cells, one chamber with compound treated cells) in 1x Low Glucose SAB. Retain 0.5 mL of cell sample for protein quantitation by BCA (Bicinchoninic acid) assay if using the mechanical dissociation approach (freeze at -20 &#176;C for measurement later).
		<ol>
			<li>Push plunger in all the way and aspirate the residual volume. Stir the cells continuously throughout the experiment at 750 rpm and 37 &#176;C for all subsequent steps. Make a mark by clicking F4 and labeling as &#34;cells&#34; when samples are loaded.</li>
			<li>Measure samples for 30 min. After signal stabilization, select a region of the change in oxygen concentration corresponding to the low glucose conditions (2.5 mM glucose) as described in 3.2.3.</li>
		</ol>
		</li>
		<li>After signal stabilization is reached, add 12.5 &#181;L of a 45% sterile glucose solution (16.7 mM final concentration) into each chamber through the titanium loading port using a syringe. Make a mark measured &#34;Glucose&#34; as described in 3.3.1 when treatment is added. Let signal stabilize and record cellular respiration until a stable oxygen flux is achieved. Select this region of the change in oxygen concentration, as described in 3.2.3. This is the 16.7 mM Glucose reading, and corresponds with stimulatory conditions7.</li>
		<li>After signal stabilization is reached add 1 &#181;L of 5mM oligomycin A (2.5 &#181;M final concentration) into each chamber through the loading port. Make a mark measured &#34;OligoA&#34; as described in 3.3.1 when treatment is added. Let signal stabilize and record cellular respiration until a stable oxygen flux is achieved. Select this area of the curve as described in 3.2.3. Oligomycin A inhibits ATP synthase and thus the only oxygen flux occurring is via leak of electrons and not oxidative phosphorylation7.</li>
		<li>After signal stabilization is reached add 1 mM FCCP in 1 &#181;L increments until a maximum respiration rate is established. This represents maximal uncoupled respiration. Between 3-4 &#181;L FCCP is sufficient (1.5-2.0 &#181;M final concentration) to induce maximal uncoupled respiration of INS-1 832/13 cells. Make a mark labeled &#34;FCCP&#34; as described in 3.3.1 when treatment is added. Let signal stabilize and record cellular respiration until a stable oxygen flux is achieved. Select this area of the curve as described in 3.2.3. FCCP is an uncoupling agent. This allows us to measure uncoupled respiration7.</li>
		<li>After signal stabilization is reached add 1 &#181;L of 5 mM Antimycin A (2.5 &#181;M final concentration) into each chamber through the loading port. Make a mark labeled &#34;AntiA&#34; as described in 3.3.1 when treatment is added. Let signal stabilize and record cellular respiration until a stable oxygen flux is achieved. Select this area of the curve as described in 3.2.3. 
		Note: Antimycin A binds cytochrome C reductase, inhibits ubiquinone oxidation, and blocks all respiration. This allows us to completely stop respiration7.</li>
	</ol>
	</li>
	<li>Measuring 832/13 beta cells for protein concentration and respiration normalization.
	<ol>
		<li>Using the 0.5 mL of sample retained at loading, measure protein sample using the BCA method13.</li>
		<li>Run each sample in triplicate, without dilution, following the manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>832/13 beta cell respiration calculations from trypsinized cells.
	<ol>
		<li>Enter the number of cells per mL use in the assay by pushing the F3 button of the respirometer analysis program. Change the units to cells/mL, enter the cellular concentration, and change medium to SAB.</li>
		<li>Select background readings to normalize the data by selecting and entering into the O2 Calibration form as described in 3.2.3.</li>
		<li>Make selections of readings from 2.5 mM Glucose, 16.7 mM Glucose, Oligomycin A, FCCP and Antimycin A as described in 3.3.1.</li>
		<li>Within the respirometer analysis program, export the readings for each chamber after entering protein concentration and selecting the appropriate average values for each treatment during the respiration measurement. This is done by clicking F2 and then pushing the &#34;copy to clipboard function&#34;. This exports the data for use in other analysis programs. Use the &#34;O2 slope neg.&#34; values for calculations.</li>
		<li>Compile data for 3-5 independent runs of vehicle treated controls and natural compounds treated cells to determine the effect on intact cellular respiration of INS-1 832/13 beta cells.</li>
	</ol>
	</li>
	<li>832/13 beta cell respiration calculations from mechanically dissociated cells.
	<ol>
		<li>Enter the protein calculations from the BCA assay by pushing the F3 button of the respirometer analysis program. Change the units to mg, enter the BCA concentration, and change medium to SAB.</li>
		<li>Select background readings to normalize the data by selecting and entering into the O2 Calibration form as described in 3.2.3.</li>
		<li>Make selections of readings from 2.5 mM Glucose, 16.7 mM Glucose, Oligomycin A, FCCP and Antimycin A as described in 3.3.1.</li>
		<li>Within the respirometer analysis program, export the readings for each chamber after entering protein concentration and selecting the appropriate average values for each treatment during the respiration measurement. This is done by clicking F2 and then pushing the &#34;copy to clipboard function&#34;. This exports the data for use in other analysis programs. Use the &#34;O2 slope neg.&#34; values for calculations.</li>
		<li>Compile data for 3-5 independent runs of vehicle treated controls and natural compounds treated cells to determine the effect on intact cellular respiration of INS-1 832/13 beta cells.</li>
	</ol>
	</li>
</ol>

<ol><li>Mueckler, M., Thorens, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+SLC2+%28GLUT%29+family+of+membrane+transporters%5BTitle%5D)+AND+%22Mol+Aspects+Med%22%5BJournal%5D)">The SLC2 (GLUT) family of membrane transporters.</a> Mol Aspects Med. 34 (2-3), 121-138 (2013).</li>
<li>Kahn, S. E., Hull, R. L., Utzschneider, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+linking+obesity+to+insulin+resistance+and+type+2+diabetes%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Mechanisms linking obesity to insulin resistance and type 2 diabetes.</a> Nature. 444 (7121), 840-846 (2006).</li>
<li>Weir, G. C., Bonner-Weir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Five+stages+of+evolving+beta-cell+dysfunction+during+progression+to+diabetes%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Five stages of evolving beta-cell dysfunction during progression to diabetes.</a> Diabetes. 53 Suppl 3, S16-S21 (2004).</li>
<li>Strat, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+by+which+cocoa+flavanols+improve+metabolic+syndrome+and+related+disorders%5BTitle%5D)+AND+%22J+Nutr+Biochem%22%5BJournal%5D)">Mechanisms by which cocoa flavanols improve metabolic syndrome and related disorders.</a> J Nutr Biochem. 35, 1-21 (2016).</li>
<li>Rowley, T. J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monomeric+cocoa+catechins+enhance+%CE%B2-cell+function+by+increasing+mitochondrial+respiration%5BTitle%5D)+AND+%22J+Nutr+Biochem%22%5BJournal%5D)">Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration.</a> J Nutr Biochem. 49, 30-41 (2017).</li>
<li>Djafarzadeh, S., Jakob, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+Respirometry+to+Assess+Mitochondrial+Function+in+Permeabilized+and+Intact+Cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells.</a> J Vis Exp. (120), (2017).</li>
<li>Reynolds, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((beta-Cell+deletion+of+Nr4a1+and+Nr4a3+nuclear+receptors+impedes+mitochondrial+respiration+and+insulin+secretion%5BTitle%5D)+AND+%22Am+J+Physiol+Endocrinol+Metab%22%5BJournal%5D)">beta-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.</a> Am J Physiol Endocrinol Metab. 311 (1), E186-E201 (2016).</li>
<li>Hals, I. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+Respiration+in+Insulin-Producing+beta-Cells%3A+General+Characteristics+and+Adaptive+Effects+of+Hypoxia%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mitochondrial Respiration in Insulin-Producing beta-Cells: General Characteristics and Adaptive Effects of Hypoxia.</a> PLoS One. 10 (9), e0138558(2015).</li>
<li>Ray, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nkx6.1-mediated+insulin+secretion+and+beta-cell+proliferation+is+dependent+on+upregulation+of+c-Fos%5BTitle%5D)+AND+%22FEBS+Lett%22%5BJournal%5D)">Nkx6.1-mediated insulin secretion and beta-cell proliferation is dependent on upregulation of c-Fos.</a> FEBS Lett. 590 (12), 1791-1803 (2016).</li>
<li>Schisler, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stimulation+of+human+and+rat+islet+beta-cell+proliferation+with+retention+of+function+by+the+homeodomain+transcription+factor+Nkx6.1%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Stimulation of human and rat islet beta-cell proliferation with retention of function by the homeodomain transcription factor Nkx6.1.</a> Mol Cell Biol. 28 (10), 3465-3476 (2008).</li>
<li>Tessem, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nkx6.1+regulates+islet+beta-cell+proliferation+via+Nr4a1+and+Nr4a3+nuclear+receptors%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Nkx6.1 regulates islet beta-cell proliferation via Nr4a1 and Nr4a3 nuclear receptors.</a> Proc Natl Acad Sci U S A. 111 (14), 5242-5247 (2014).</li>
<li>Hobson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aurora+Kinase+A+is+critical+for+the+Nkx6.1+mediated+beta-cell+proliferation+pathway%5BTitle%5D)+AND+%22Islets%22%5BJournal%5D)">Aurora Kinase A is critical for the Nkx6.1 mediated beta-cell proliferation pathway.</a> Islets. 7 (1), e1027854(2015).</li>
<li>Draney, C., Hobson, A. E., Grover, S. G., Jack, B. O., Tessem, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cdk5r1+Overexpression+Induces+Primary+beta-Cell+Proliferation%5BTitle%5D)+AND+%22J+Diabetes+Res%22%5BJournal%5D)">Cdk5r1 Overexpression Induces Primary beta-Cell Proliferation.</a> J Diabetes Res. 2016, 6375804(2016).</li>
<li>Rouse, M., Younes, A., Egan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resveratrol+and+curcumin+enhance+pancreatic+beta-cell+function+by+inhibiting+phosphodiesterase+activity%5BTitle%5D)+AND+%22J+Endocrinol%22%5BJournal%5D)">Resveratrol and curcumin enhance pancreatic beta-cell function by inhibiting phosphodiesterase activity.</a> J Endocrinol. 223 (2), 107-117 (2014).</li>
<li>Ma, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+reduces+beta-cell+mitochondria+and+induces+distinct+morphological+abnormalities%2C+which+are+reproducible+by+high+glucose+in+vitro+with+attendant+dysfunction%5BTitle%5D)+AND+%22Islets%22%5BJournal%5D)">Diabetes reduces beta-cell mitochondria and induces distinct morphological abnormalities, which are reproducible by high glucose in vitro with attendant dysfunction.</a> Islets. 4 (3), 233-242 (2012).</li>
<li>Chavanelle, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+high-intensity+interval+training+and+moderate-intensity+continuous+training+on+glycaemic+control+and+skeletal+muscle+mitochondrial+function+in+db%2Fdb+mice%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Effects of high-intensity interval training and moderate-intensity continuous training on glycaemic control and skeletal muscle mitochondrial function in db/db mice.</a> Sci Rep. 7 (1), 204(2017).</li>
<li>Jelenik, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+Insulin+Resistance+in+Primary+and+Secondary+Nonalcoholic+Fatty+Liver%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Mechanisms of Insulin Resistance in Primary and Secondary Nonalcoholic Fatty Liver.</a> Diabetes. 66 (8), 2241-2253 (2017).</li>
<li>Spacek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose-stimulated+insulin+secretion+of+insulinoma+INS-1E+cells+is+associated+with+elevation+of+both+respiration+and+mitochondrial+membrane+potential%5BTitle%5D)+AND+%22Int+J+Biochem+Cell+Biol%22%5BJournal%5D)">Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential.</a> Int J Biochem Cell Biol. 40 (8), 1522-1535 (2008).</li>
<li>Jezek, J., Dlaskova, A., Zelenka, J., Jaburek, M., Jezek, P. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623989/">H2O2-Activated Mitochondrial Phospholipase iPLAgamma Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic beta-Cells.</a> Antioxid Redox Signal. 23 (12), 958-972 (2015).</li>
<li>Johnson, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppressed+insulin+signaling+and+increased+apoptosis+in+CD38-null+islets%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Suppressed insulin signaling and increased apoptosis in CD38-null islets.</a> Diabetes. 55 (10), 2737-2746 (2006).</li>
</ol>

The authors have nothing to disclose.

The objective of this protocol is to use high-resolution respirometry to measure respiratory rates in intact pancreatic beta cells. This method allows the measurement of the beta cell response to increased glucose levels. The protocol also allows for the pretreatment with various compounds, as demonstrated in this protocol with the naturally occurring monomeric epicatechin or curcumin4,5. Treatment with various other compounds cou...

The primary purpose of the pancreatic beta cell is to maintain system normoglycemia through glucose-stimulated insulin secretion. The beta cells sense physiological changes in circulating glucose largely due to the low affinity, high capacity glucose transporter GLUT2 (Glucose Transporter 2, Km 16.7 mM)1. As circulatory glucose levels rise, this high-capacity low-affinity transporter facilitates a proportional increase in intracellular glucose within the beta cell. Glucose is metabolized through glycolysis, the TCA cycle (tricarboxylic acid cycle) and mitochondrial respiration resulting in elevated cellular ATP (adenosine triphosphate) levels. The elevated ATP concentration blocks the ATP sensitive K+ channels, resulting in membrane depolarization. Membrane depolarization causes the opening of voltage gated Ca2+ channels and subsequent release of vesicle bound insulin granules2. Beta cell dysfunction is a hallmark of Type 2 Diabetes (T2D), and results in decreased and poorly controlled insulin secretion and ultimately beta cell death3. Mechanisms that maintain or improve beta cell function could be used as a treatment for T2D.
Studies have demonstrated the beneficial effects of naturally occurring plant-based compounds on the pancreatic beta cell4. These compounds may have their effect through increasing beta cell proliferation, survival, or glucose-stimulated insulin secretion. As an example, recent studies have demonstrated that monomeric epicatechin enhances glucose-stimulated insulin secretion through increasing mitochondrial respiration and increasing cellular ATP levels5. Therefore, understanding how these compounds can increase functional beta cell mass is important to leverage these compounds as potential therapeutics.
Cellular respiration can be measured through a number of tools. Use of a high-resolution respirometer allows for titration of chemical modulators to a permeabilized or intact cell population6. This tool permits the addition of various compounds, at different concentrations, thus giving a wide array of information.
Given the intimate connection between glucose metabolism and beta cell function, measurements of cellular respiration are critical. Measurements of cellular respiration can be done using either permeabilized or intact beta cells, with each having its own set of benefits and drawbacks7,8. While permeabilization of beta cells allows one to measure different aspects of the electron transport chain, it does so without regards to the mechanism for inducing respiration in the beta cell, glucose uptake and metabolism. Therefore, use of unpermeabilized beta cell respiration is a very useful technique to determine the beta cells response to various glucose levels, using oxygen consumption as the readout.
The purpose of this technique is to measure oxygen consumption in intact INS-1 derived 832/13 beta cells. This technique allows us to determine the response of the beta cells to unstimulatory glucose conditions (2.5 mM glucose) as well as stimulatory glucose conditions (16.7 mM glucose). While the unpermeabilized cells do not allow us to individually test complex I, II or III of the electron transport chain, the technique does permit measurements dealing with complex IV inhibition (Oligomycin A), uncoupled respiration (FCCP-Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone), and completely inhibited respiration (Antimycin A). This study demonstrates the efficacy of measuring respiration in intact unpermeabilized pancreatic beta cells, as well as the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration.

<table><tbody><tr><td>O2k Core: Oxygraph-2k</td><td>Oroboros Instruments</td><td>10000-02</td><td>Instrument for high-resolution respirometry</td></tr><tr><td>DatLab 6 Program</td><td>Oroboros Instruments</td><td>27141-01</td><td>Computer program for analysing high-reolution respirometry</td></tr><tr><td>INS-1 832/13 cell line</td><td>Duke University Medical Center</td><td>NONE</td><td>Beta cell line, gift from Dr. Christopher Newgard</td></tr><tr><td>Curcumin</td><td>Sigma</td><td>C7727</td><td>Pre-treatment of beta cells</td></tr><tr><td>Cocoa epicatechin monomer</td><td>Virginia Polytechnic Institute and State University</td><td>NONE</td><td>Pre-treatment of beta cells, gift from Dr. Andrew Neilson</td></tr><tr><td>Trypsin</td><td>Sigma</td><td>T4049</td><td>For cell culture</td></tr><tr><td>RPMI-1640</td><td>Sigma</td><td>R8758</td><td>832/13 beta cell media</td></tr><tr><td>Fetal Bovine Serum</td><td>Hyclone</td><td>SH30071.03</td><td>832/13 beta cell media component</td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma</td><td>P4458</td><td>832/13 beta cell media component</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3662</td><td>832/13 beta cell media component/ 1x SAB Buffer</td></tr><tr><td>Glutamine</td><td>Caisson</td><td>GLL01</td><td>832/13 beta cell media component</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>S8636</td><td>832/13 beta cell media component</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma</td><td>M3148</td><td>832/13 beta cell media component</td></tr><tr><td>NaCl</td><td>Fisher</td><td>S271</td><td>Component of 10x SAB buffer</td></tr><tr><td>KCl</td><td>Sigma</td><td>P9541</td><td>Component of 10x SAB buffer</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P5655</td><td>Component of 10x SAB buffer</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>M2643</td><td>Component of 10x SAB buffer</td></tr><tr><td>D-(+)-Glucose Solution</td><td>Sigma</td><td>G8769</td><td>Component of 1x SAB buffer, chemical for respiration assay</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>C5670</td><td>Component of 1x SAB buffer</td></tr><tr><td>35% BSA Solution</td><td>Sigma</td><td>A7979</td><td>Component of 1x SAB buffer</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td>Component of 1x SAB buffer</td></tr><tr><td>200 proof ethanol</td><td>Sigma</td><td>459844</td><td>Washing Oxygraph O2k Chambers</td></tr><tr><td>Oligomycin A</td><td>Sigma</td><td>O4876</td><td>Chemicals for respiration assay</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C2920</td><td>Chemicals for respiration assay</td></tr><tr><td>Anitmycin A</td><td>Sigma</td><td>A8674</td><td>Chemicals for respiration assay</td></tr><tr><td>BCA Protein Kit</td><td>Thermo Fisher</td><td>23225</td><td>For determining protein concentration</td></tr></tbody></table>

high-resolution respirometry to measure mitochondrial function of intact beta cells in the presence of natural compounds

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role in the body by controlling blood glucose levels through insulin secretion in response to elevated glucose concentrations. Insulin secretion is controlled by glucose metabolism and mitochondrial respiration. Therefore, measuring intact beta cell respiration is essential to be able to improve beta cell function as a treatment for diabetes. Using intact 832/13 INS-1 derived beta cells we can measure the effect of increasing glucose concentration on cellular respiration. This protocol allows us to measure beta cell respiration in the presence or absence of various compounds, allowing one to determine the effect of given compounds on intact cell respiration. Here we demonstrate the effect of two naturally occurring compounds, monomeric epicatechin and curcumin, on beta cell respiration under the presence of low (2.5 mM) or high glucose (16.7 mM) conditions. This technique can be used to determine the effect of various compounds on intact beta cell respiration in the presence of differing glucose concentrations.

INS-1 832/13 beta cells that are prepared and harvested as described in the protocol will demonstrate modulation in oxygen consumption based on the various chemical interventions (Figure 1A). An increase in respiration will be observed when the glucose concentration is increased to 16.7 mM Glucose (Figure 1B). Respiration will decrease when the intact cells are treated with Oligomycin A. This respiration is known as LEAK, which is defined as the basal, nonphosph...

The goal of this protocol is to measure the effect of glucose-mediated changes to mitochondrial respiration in the presence of natural compounds on intact 832/13 beta cells using high-resolution respirometry.

High-resolution respirometry allows for the measurement of oxygen consumption of isolated mitochondria, cells and tissues. Beta cells play a critical role ...

high-resolution-respirometry-to-measure-mitochondrial-function-intact

Research

JoVE Journal

Biology

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes' (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

Here, a quadriceps muscle specimen is taken from an anaesthetized pig and mitochondria are isolated by differential centrifugation. Then, the respiratory rates of mitochondrial respiratory chain complexes I, II and IV are determined using high-resolution respirometry.

Isolation of Intact Mitochondria from Skeletal Muscle by Differential Centrifugation for High-resolution Respirometry Measurements

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry coupled to fluorescence sensors determines mitochondrial oxygen consumption and reactive oxygen species (ROS) generation. The present protocol describes a technique to assess mitochondrial respiratory rates and ROS production in the permeabilized sciatic nerve.

Assessing Mitochondrial Function in Sciatic Nerve by High-Resolution Respirometry

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Here, we describe a comprehensive method for measuring mitochondrial oxidative phosphorylation in fresh permeabilized skeletal muscle fibers from either human or mouse muscle. This method allows for the real-time quantification of mitochondrial respiration and the assessment of fuel preference and metabolic flexibility while preserving existing mitochondrial networks and membrane integrity.

Measurement of Mitochondrial Respiration in Human and Mouse Skeletal Muscle Fibers by High-Resolution Respirometry

Methods for studying mitochondrial bioenergetics under physiologically relevant substrate concentrations in immune cells are limited. We provide a detailed protocol that uses high-resolution fluorespirometry to assess changes in the response of the mitochondrial membrane potential to energy demand in human T-cells, monocytes, and peripheral mononuclear cells.

High-Resolution Fluorespirometry to Assess Dynamic Changes in Mitochondrial Membrane Potential in Human Immune Cells

Here, we show that the 0.5-mL chamber for high-resolution respirometry shows results that are consistent with the 2.0-mL chamber if appropriate instrumental O2 background correction is applied. It requires less sample, which is advantageous for studies with limited availability or low respiratory capacities.

High-Resolution Respirometry in a Small-Volume Chamber

Learn high-resolution respirometry to measure mitochondrial function in intact pancreatic beta cells using INS-1-derived 832/13 cells. This method evaluates natural compound effects on respiration under insulin stimulatory conditions for diabetes research.

High-resolution Respirometry to Measure Mitochondrial Function of Intact Beta Cells in the Presence of Natural Compounds

Explore More Videos