Name: analysis of non-human primate pancreatic islet oxygen consumption
Uploaded: 2019-12-18T15:00:00.000+00:00
Duration: 7 min 12 s
Description: Watch this Scientific Journal Video about Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption at JoVE.com

The authors would like to acknowledge the Vanderbilt High Throughput Screening Core for the use of their facilities, Agilent Biotechnologies, Dr. Paul Kievit (Oregon Health and Science University) for non-human primate islet isolations, and Eric Donahue (Vanderbilt University) for assistance with Figure 1. J.M.E. was supported by NIGMS of the National Institutes of Health under award number T32GM007347. M.G. was supported by the NIH/NIDDK (R24DK090964-06) and the Department of Veterans Affairs (BX003744).

1. Preparation of Microplate and Sensor Cartridge on the Day Prior to Running the Assay
Islets were isolated from three year old Japanese macaques as previously described13. This method is very similar to that used to isolate human islets from cadaver donors, but differs from mice, in which pancreata are often inflated with collagenase solution while the animal is under sedation and prior to organ removal. Islet retrieval was conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Oregon National Primate Research Center (ONPRC) and Oregon Health and Science University and were approved by the ONPRC IACUC. The ONPRC abides by the Animal Welfare Act and Regulations enforced by the United States Department of Agriculture (USDA) and the Public Health Service Policy on Humane Care and Use of Laboratory Animals in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
<ol>
	<li>Preparation of Spheroid Microplate
	<ol>
		<li>Prepare 3 mL of a 0.1M solution of sodium bicarbonate. Filter sterilize the solution. We utilized a 0.45 &#181;m filter (Table of Materials).</li>
		<li>In a cell culture hood, add 200 &#181;L of cell and tissue adhesive (Table of Materials) to 2.8 mL of 0.1M sodium bicarbonate. Then, add 20 &#181;L of this solution to the bottom of each well of the 96-well spheroid microplate (Table of Materials). Ensure that air bubbles are removed from the pipet tip and only the bottom of the plate is covered. Coating of the microplate prevents islets from moving up the sides of the wells when drugs are added and mixed into the well during the assay. 
		NOTE: It is only necessary to coat as many wells as will be used for islets when the assay is run. If only a few wells will be used, the cell adhesive solution can be scaled down appropriately. The cell adhesive used in the assay is a formulated protein solution extracted from the marine mussel. Alternatively, microplate wells can be coated with 20 &#181;L of 100 &#181;g/mL poly-D-lysine.</li>
		<li>Incubate the plate in a 37 &#176;C, non-CO2 incubator for one hour.</li>
		<li>In a sterile environment, aspirate cell adhesive solution from the plate. Wash each well two times with 400 &#181;L of 37 &#176;C sterile water using a multichannel pipet. Allow to air dry.</li>
		<li>After 30-40 minutes, cover the plate and store overnight at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Hydration of the Sensor Cartridge
	<ol>
		<li>In a sterile environment, open the sensor cartridge package (Table of Materials) and remove the contents. Place the sensor cartridge upside down next to the utility plate.</li>
		<li>Fill each well of the utility plate with 200 &#181;L sterile water and lower the sensor cartridge back into the utility plate. Place assembled sensor cartridge and utility plate in a 37 &#176;C, non-CO2 incubator overnight.</li>
	</ol>
	</li>
	<li>Preparation of Calibrant
	<ol>
		<li>Aliquot 25 mL of calibrant (Table of Materials) into a 50 mL conical tube. Place tube in a 37 &#176;C, non-CO2 incubator overnight.</li>
	</ol>
	</li>
</ol>
2. Protocol for Media Preparation, Loading of Islets, and Loading of Sensor Cartridge on the Day of Assay
<ol>
	<li>Preparation of media
	<ol>
		<li>To 48.5 mL of base media (minimal DMEM, see Table of Materials), add 500 &#181;L each of 1 mM sodium pyruvate and 2 mM glutamine. Additionally, add 496 &#181;L of 200 mg/ml glucose (final concentration of glucose in the media is 5.5 mM). 
		NOTE: Baseline glucose concentration was kept constant for these experiments. However, glucose can also be used to stimulate respiration in addition to the pharmacologic manipulations described blow10.</li>
		<li>Confirm that the pH is 7.4 +/- 0.1, adjusting if necessary.</li>
	</ol>
	</li>
	<li>Preparation of sensor cartridge
	<ol>
		<li>Take sensor cartridge out of incubator, remove sensor cartridge lid, and dump out water from wells. Place 200 mL of pre-warmed calibrant into each well and replace sensor cartridge lid.</li>
		<li>Place sensor cartridge back in incubator for about 1 hour (until needed).</li>
	</ol>
	</li>
	<li>Preparation of drugs for mitochondrial stress assay
	<ol>
		<li>Open the stress test kit (Table of Materials) and remove the contents. Make up stock and working solutions for Oligomycin, Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and Rotenone/Antimycin A (AA) as follows.
		<ol>
			<li>Oligomycin: add 630 &#181;L of assembled media to tube to make 100 &#181;M stock. Then, dilute to 45 &#181;M with media to obtain port concentration (final well concentration after injection will be 4.5 &#181;M)</li>
			<li>FCCP: add 720 &#181;L of assembled media to tube to make 100 &#181;M stock. Dilute to 10 &#181;M with media to obtain port concentration (final well concentration will be 1 &#181;M) 
			NOTE: BSA and/or FBS should not be added to media, as this can affect the action of mitochondrial uncouplers14.</li>
			<li>Rotenone/AA: add 540 &#181;L of assembled media to tube to make 50 &#181;M stock. Dilute to 25 &#181;M with media to obtain port concentration (final well concentration will be 2.5 &#181;M) 
			NOTE: The above concentrations have produced consistent results in our hands. Additional FCCP led to no further increase in respiration. However, drug concentrations may need to be adjusted depending on islet size, and especially with islets from different species or non-islet spheroids. For reference, the average diameter of a non-human primate islet is about 150 &#181;m15.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of spheroid plate and transfer of islets
	<ol>
		<li>Hand-pick pancreatic islets into a 60 mm x 15 mm cell culture dish containing assembled media to obtain near 100% purity. 
		NOTE: Islets should appear rounded, with a clearly defined periphery, and appear denser than exocrine tissue contaminants. Avoid islets that appear damaged or frayed. In this experiment, islet size was not directly measured, although we attempted to pick islets of approximately equivalent size for each well and sample.</li>
		<li>Load each well of spheroid microplate with 175 &#181;L of assembled media using a multichannel pipette.</li>
		<li>Using a P20 pipette set to 15 &#181;L, aspirate 15 islets from cell culture dish. Islets should be visible in the pipette tip to the naked eye.</li>
		<li>To transfer islets to spheroid microplate, lower pipette tip to the bottom of the well, barely lift up, and slowly pipette out a very small volume (~5 &#181;L). Confirm that all islets have left the pipette tip. Each well should receive 15 islets. Occasionally check spheroid microplate under the microscope to verify that islets are at the bottom of each well rather than stuck to the sides. 
		NOTE: Avoid loading the corner wells with islets. Oxygen and pH flux out of the plastic can occur during the assay, and this is exacerbated in the four corner wells.</li>
		<li>Once all islets have been transferred to the spheroid microplate, incubate the microplate in a 37 &#176;C, non-CO2 incubator while completing the steps below.</li>
	</ol>
	</li>
	<li>Loading the sensor cartridge and running the assay
	<ol>
		<li>Remove sensor cartridge from incubator. Ports A-C for each well must be loaded with either drug or media. Wells containing islets and the four corner wells should be loaded with drug. Non-corner wells without islets should be loaded with media. Port D can be left empty. Load port A (top left of each sensor) with 20 &#181;L of oligomycin or media. Load port B (top right) with 22 &#181;L of FCCP or media. Load port C (bottom left) with 25 &#181;L of Rotenone/AA or media.</li>
		<li>Program an extracellular flux analyzer assay for a 30 minute baseline respiration period (5 cycles of 3 minute mix, 3 minute measure), 42 minute oligomycin measurement period (7 cycles of 3 minute mix, 3 minute measure), 30 minutes FCCP (5 cycles of 3 minute mix, 3 minute measure), and 30 minutes Rotenone/AA (5 cycles of 3 minute mix, 3 minute measure).</li>
		<li>Run the assay and follow instructions on screen for calibrating the sensor and inserting the microplate.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Mulder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcribing+&#946;-cell+mitochondria+in+health+and+disease.">Transcribing &#946;-cell mitochondria in health and disease.</a> Molecular Metabolism. 6 (9), 1040-1051 (2017).</li><li>Maechler, P., Wollheim, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+signals+in+glucose-stimulated+insulin+secretion+in+the+beta+cell.">Mitochondrial signals in glucose-stimulated insulin secretion in the beta cell.</a> The Journal of Physiology. 529 (Pt 1), 49-56 (2000).</li><li>Curry, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon+Potentiation+of+Insulin+Secretion+by+the+Perfused+Rat+Pancreas.">Glucagon Potentiation of Insulin Secretion by the Perfused Rat Pancreas.</a> Diabetes. 19 (6), 420 (1970).</li><li>Song, G., Pacini, G., Ahr&#233;n, B., D'Argenio, D. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucagon+Increases+Insulin+Levels+by+Stimulating+Insulin+Secretion+Without+Effect+on+Insulin+Clearance+in+Mice.">Glucagon Increases Insulin Levels by Stimulating Insulin Secretion Without Effect on Insulin Clearance in Mice.</a> Peptides. 88, 74-79 (2017).</li><li>Vergari, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+inhibits+glucagon+release+by+SGLT2-induced+stimulation+of+somatostatin+secretion.">Insulin inhibits glucagon release by SGLT2-induced stimulation of somatostatin secretion.</a> Nature Communications. 10 (1), 139 (2019).</li><li>Watts, M., Ha, J., Kimchi, O., Sherman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+glucagon+secretion:+the+&#946;/&#945;/&#948;+model.">Paracrine regulation of glucagon secretion: the &#946;/&#945;/&#948; model.</a> American Journal of Physiology--Endocrinology and Metabolism. 310 (8), E597-E611 (2016).</li><li>Sweet, I. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+measurement+of+oxygen+consumption+by+pancreatic+islets.">Continuous measurement of oxygen consumption by pancreatic islets.</a> Diabetes Technology &amp; Therapeutics. 4 (5), 661-672 (2002).</li><li>. Agilent Seahorse XF Instruments Overview and Selection Guide Available from: <a target="_blank" href="https://www.agilent.com/en/products/cell-analysis/seahorse-xf-instruments-selection-guide">https://www.agilent.com/en/products/cell-analysis/seahorse-xf-instruments-selection-guide</a> (2019)</li><li>Wikstrom, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+high-throughput+assay+for+islet+respiration+reveals+uncoupling+of+rodent+and+human+islets.">A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.</a> PLoS One. 7 (5), e33023 (2012).</li><li>Taddeo, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individual+islet+respirometry+reveals+functional+diversity+within+the+islet+population+of+mice+and+human+donors.">Individual islet respirometry reveals functional diversity within the islet population of mice and human donors.</a> Molecular Metabolism. 16, 150-159 (2018).</li><li>Conrad, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MAFB+transcription+factor+impacts+islet+alpha-cell+function+in+rodents+and+represents+a+unique+signature+of+primate+islet+beta-cells.">The MAFB transcription factor impacts islet alpha-cell function in rodents and represents a unique signature of primate islet beta-cells.</a> American Journal of Physiology--Endocrinology and Metabolism. 310 (1), E91-E102 (2016).</li><li>Steiner, D. J., Kim, A., Miller, K., Hara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+islet+plasticity:+interspecies+comparison+of+islet+architecture+and+composition.">Pancreatic islet plasticity: interspecies comparison of islet architecture and composition.</a> Islets. 2 (3), 135-145 (2010).</li><li>Elsakr, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Western-style+diet+affects+offspring+islet+composition+and+function+in+a+non-human+primate+model+of+maternal+over-nutrition.">Maternal Western-style diet affects offspring islet composition and function in a non-human primate model of maternal over-nutrition.</a> Molecular Metabolism. , (2019).</li><li>Soutar, M. P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FBS/BSA+media+concentration+determines+CCCP's+ability+to+depolarize+mitochondria+and+activate+PINK1-PRKN+mitophagy.">FBS/BSA media concentration determines CCCP's ability to depolarize mitochondria and activate PINK1-PRKN mitophagy.</a> Autophagy. , 1-10 (2019).</li><li>Hirshberg, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+Islet+Transplantation+Using+the+Nonhuman+Primate+(Rhesus)+Model+Predicts+That+the+Portal+Vein+Is+Superior+to+the+Celiac+Artery+as+the+Islet+Infusion+Site.">Pancreatic Islet Transplantation Using the Nonhuman Primate (Rhesus) Model Predicts That the Portal Vein Is Superior to the Celiac Artery as the Islet Infusion Site.</a> Diabetes. 51 (7), 2135-2140 (2002).</li><li>Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+and+interpretation+of+microplate-based+oxygen+consumption+and+pH+data.">Analysis and interpretation of microplate-based oxygen consumption and pH data.</a> Methods in Enzymology. 547, 309-354 (2014).</li><li>Papas, K. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+Oxygen+Consumption+Rate+(OCR)+Dose+Predicts+Insulin+Independence+in+Clinical+Islet+Autotransplantation.">Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.</a> PLoS One. 10 (8), e0134428 (2015).</li></ol>

The authors have nothing to disclose.

The study of islet oxygen consumption has previously been hampered by the spherical shape of islets, their lack of adherence to culture surfaces, and the number of islets required per well. In this protocol, we highlight the efficacy of the 96-well spheroid microplate for measuring islet oxygen consumption on a small number of islets and demonstrate a technique for handling and loading islets which is technically feasible and produces consistent results.
In order for islets to adhere to the bo...

In order to maintain normal blood glucose levels, the pancreatic &#946; cell must sense elevations in glucose and secrete insulin accordingly. The coupling of insulin secretion with glucose levels is directly linked to glucose metabolism and the production of ATP through mitochondrial oxidative phosphorylation. Thus, mitochondria play a critical role in stimulus-secretion coupling1. Assessing &#946;-cell mitochondrial function can reveal defects that lead to impaired insulin secretion. The secretion of glucagon by pancreatic &#945; cells is also closely tied to mitochondrial function2. Although immortalized islet cell lines have proven useful for some types of assays, the physiology of these cells does not accurately recapitulate whole islet function, as illustrated by the potentiation of insulin secretion by glucagon3,4 and the inhibition of glucagon secretion by insulin/somatostatin5,6 in intact islets. This demonstrates the need for measuring oxygen consumption using whole, intact islets.
Techniques for the measurement of islet cell respirometry have evolved over time, from the use of oxygen-sensitive fluorescent dyes7 to solid-state sensors that directly measure oxygen consumption8. Initially designed for monolayer, adherent cells, commonly used cell culture plate systems have proven to be ineffective for pancreatic islets. As islets do not naturally adhere to the wells, they are prone to being pushed to the periphery of the culture well resulting in inaccurate measurement of oxygen consumption9. To combat this problem, specialized 24-well plates with a central depression that could contain islets were developed9. However, the 24-well plate system was limited by the large number of islets required (50-80 per well) and the number of conditions that could be tested simultaneously10. The recent development of 96-well microplates designed specifically for extracellular flux analysis in spheroids has overcome these barriers, enabling the measurement of islet respirometry with 20 or fewer islets per well10.
Here, we demonstrate the use of this system to measure oxygen consumption in islets from the Japanese macaque (Macaca fuscata), an animal model with similar islet biology to humans11,12. In this protocol, 15 macaque islets are analyzed per well. In our hands, 15 islets per well produced higher baseline oxygen consumption than fewer islets, with robust activation and repression of respiration in response to pharmacologic manipulation. We highlight the steps to prepare for the assay, an effective method for consistent loading of islets at the center of each well, and common challenges when performing this assay.

<table><tbody><tr><td>Cell culture dish, 60 mm X 15 mm style</td><td>Corning</td><td>430166</td><td></td></tr><tr><td>Cell-Tak Cell and Tissue Adhesive</td><td>Corning</td><td>354240</td><td></td></tr><tr><td>Conical tube, 50 mL</td><td>Falcon</td><td>352070</td><td></td></tr><tr><td>Dextrose anhydrous</td><td>Fisher Scientific</td><td>BP350-1</td><td>For glucose solution, 200 mg/ml, sterile filetered</td></tr><tr><td>Disposable reservoirs (sterile), 25 ML</td><td>Vistalab</td><td>3054-1033</td><td>for loading multichannel pipet</td></tr><tr><td>EZFlow Sterile 0.45 &amp;mu;m PES Syringe Filter, 13 mm</td><td>Foxx Life Sciences</td><td>371-3115-OEM</td><td></td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td>200 mM (100x)</td></tr><tr><td>Multichannel pipette tips</td><td>ThermoFisher Scientific</td><td>94410810</td><td></td></tr><tr><td>Multichannel pipette, 15-1250 &amp;mu;L</td><td>ThermoFisher Scientific</td><td>4672100BT</td><td>Recommended</td></tr><tr><td>P20, P200, and P1000 pipettes</td><td>Eppendorf</td><td>2231000602</td><td></td></tr><tr><td>pH Probe</td><td>Hanna Instruments</td><td>HI2210-01</td><td></td></tr><tr><td>Pipette tips, 20 &amp;mu;L, 200 &amp;mu;L, 1000 &amp;mu;L</td><td>Olympus</td><td>24-404, 24-412, 24-430</td><td></td></tr><tr><td>Seahorse XF Base Media</td><td>Agilent</td><td>103334-100</td><td></td></tr><tr><td>Seahorse XF Cell Mito Stress Test Kit</td><td>Agilent</td><td>103015-100</td><td>Includes Oligomycin, FCCP, and Rotenone/Antimycin A</td></tr><tr><td>Seahorse XFe96 Analyzer</td><td>Agilent</td><td>S7800B</td><td>Including prep station with 37 &amp;deg;C non-CO2 incubator</td></tr><tr><td>Seahorse XFe96 Spheroid Fluxpak Mini</td><td>Agilent</td><td>102905-100</td><td>Includes sensor cartridge, spheroid microplate, and calibrant</td></tr><tr><td>Sodium bicarbonate</td><td>Fisher Scientific</td><td>BP328-500</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Gibco</td><td>11360-070</td><td>100 mM (100x)</td></tr><tr><td>Stereo Microscope</td><td>Olympus</td><td>SZX9</td><td></td></tr><tr><td>Syringe (sterile), 5 mL</td><td>BD</td><td>309603</td><td>For sterile filtration</td></tr><tr><td>Water (sterile)</td><td>Sigma</td><td>W3500-500mL</td><td></td></tr></tbody></table>

analysis of non-human primate pancreatic islet oxygen consumption

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate the measurement of islet oxygen consumption using a 96-well microplate designed for the measurement of oxygen consumption in spheroids. In this assay, spheroid microplates are coated with a cell and tissue adhesive on the day prior to the assay. We utilize a small volume of adhesive solution to encourage islet adherence to only the bottom of the well. On the day of the assay, 15 islets are loaded directly into the base of each well using a technique that ensures optimal positioning of islets and accurate measurement of oxygen consumption. Various aspects of mitochondrial respiration are probed pharmacologically in non-human primate islets, including ATP-dependent respiration, maximal respiration, and proton leak. This method allows for consistent, reproducible results using only a small number of islets per well. It can theoretically be applied to any cultured spheroids of similar size.

To load islets into microplate, 15 islets should be aspirated in 15 &#181;L of media, as shown in Figure 1A. Islets will naturally settle toward the bottom of the pipet tip within a few seconds. Then, the pipet tip is lowered to the bottom of the well. The tip is very slightly lifted, and a small volume (about 5 &#181;L) is pipetted out along with the islets. This technique results in consistent placement of islet at the bottom of the micropl...

Watch this Scientific Journal Video about Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption at JoVE.com

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

This protocol demonstrates the accurate and reproducible measurement of oxygen consumption in non-human primate pancreatic islets. The islet loading techniques and coating of the microplate provide a framework for efficient measurement of respiration in other types of cultured spheroids.

The measurement of oxygen consumption in spheroid clusters of cells, such as ex vivo pancreatic islets, has historically been challenging. We demonstrate ...

analysis-of-non-human-primate-pancreatic-islet-oxygen-consumption

Research

JoVE Journal

Developmental Biology

5.4K Views.  Vanderbilt University. Deze methode maakt het mogelijk om het zuurstofverbruik te kwantificeren door kleine aantallen eilandjes van relevante soorten, zoals niet-menselijke primaten en mensen, waardoor de mitochondriale functie kan worden beoordeeld. Deze techniek kan worden uitgevoerd met kleine aantallen primaire eilandjes, het verhogen van de technische replica's, waardoor het testen van meerdere voorwaarden, en het verbeteren van de strengheid en reproduceerbaarheid van de test. Deze techniek beoordeelt mitocho...

Video: Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological states of islet hypoxia, especially in transplant environments. Standard hypoxic chamber techniques cannot modulate both stimulations at the same time nor provide real-time monitoring of glucose stimulus-secretion coupling factors. To address these difficulties, we applied a multilayered microfluidic technique to integrate both aqueous and gas phase modulations via a diffusion membrane. This creates a stimulation sandwich around the microscaled islets within the transparent polydimethylsiloxane (PDMS) device, enabling monitoring of the aforementioned coupling factors via fluorescence microscopy. Additionally, the gas input is controlled by a pair of microdispensers, providing quantitative, sub-minute modulations of oxygen between 0-21%. This intermittent hypoxia is applied to investigate a new phenomenon of islet preconditioning. Moreover, armed with multimodal microscopy, we were able to look at detailed calcium and KATP channel dynamics during these hypoxic events. We envision microfluidic hypoxia, especially this simultaneous dual phase technique, as a valuable tool in studying islets as well as many ex vivo tissues.

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Simultaneous oxygenation and monitoring of glucose stimulus-secretion coupling factors in a single technique is critical for modeling pathophysiological ...

Bioengineering

Characterization of Metabolic Status in Nonhuman Primates with the Intravenous Glucose Tolerance Test

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical profiles, inclusive of blood glucose levels in both the fed and fasted state, HbA1c, insulin levels, clinical history of diet, body composition, and body weight status, an assessment of normal and abnormal glycemic control can be made. Interpretation of an IVGTT is done through measurement of changes in glucose and insulin levels over time in relation to the dextrose challenge. Critical components to be considered are: peak glucose and insulin levels reached in relation to T0 (end of glucose infusion), the glucose clearance rate K derived from the slope of rapid glucose clearance in the first 20 min (T1 to T20), the time to return to glucose baseline, and the area under the curve (AUC). These IVGTT measures will show characteristic changes as glucose homeostasis moves from a healthy to a diseased metabolic state5. Herein we will describe the characterization of nonhuman primates (Rhesus and Cynomolgus macaques), which are the most relevant animal model of Type II diabetes (T2D) in humans and the IVGTT and clinical profiles of these animals from a lean healthy, to obese dysmetabolic, and T2D state 8, 10, 11.

The goal of this protocol is to present a standard method to perform intravenous glucose tolerance tests (IVGTTs) to assess glycemic control in nonhuman primates and assess their metabolic status from healthy to dysmetabolic.

The intravenous glucose tolerance test (IVGTT) plays a key role in the characterization of glucose homeostasis. When taken together with serum biochemical ...

Medicine

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

Biochemistry

Mitochondrial Functional Assessment in Brush Lizard Liver Mitochondria

Assessment of Mitochondrial Oxygen Consumption Using a Plate Reader-based Fluorescent Assay

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of metabolic pathways. Therefore, experimentally assessing mitochondrial functionality can provide insight into variations among different populations or disease states. Additionally, it is valuable to assess whether isolated mitochondria are healthy enough to proceed with experiments. One characteristic often used to compare mitochondrial function in different samples is the rate of oxygen consumption. Oxygen consumption and subsequent calculation of the respiratory control ratio in either intact cells or mitochondria isolated from tissue can serve all three purposes. Using mitochondria isolated from the livers of brush lizards in conjunction with a phosphorescent probe that is sensitive to the fluctuations in oxygen concentration of a solution, we measured oxygen consumption using a fluorescent plate reader. This method is not only quick and efficient but also can be conducted with a small amount of mitochondria and without the need for specialized equipment. The step-by-step protocol described here increases the accessibility of mitochondrial functional assessment to researchers.

Assessment of oxygen consumption provides integral information about mitochondrial function. Using a phosphorescent probe with a fluorescent plate reader, accurate and reproducible data can be obtained easily without specialized equipment. This assay enables any lab to measure the oxygen consumption of isolated mitochondria and calculate respiratory control ratios.

Mitochondria serve many important functions, including cellular respiration, ATP production, controlling apoptosis, and acting as a central hub of ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Immunology and Infection

From RNA to Metabolites: Multi-Omic Profiling of Non-Human Primate Fetal Islets

Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics

One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.

Author Spotlight: Multi-Layered Approach to Understand Postnatal Functions of Pancreatic Islets in Non-Human Primates

This protocol describes procedures to isolate high-quality nuclei from frozen non-human primate pancreatic islets for use in single-nucleus simultaneous RNA sequencing and ATAC sequencing while preserving the bulk cytosolic fraction for metabolomic analyses from the same samples.

One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson&#39;s disease (PD), Huntington&#39;s disease, and Alzheimer&#39;s disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington&#39;s disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington&#39;s disease.

Oxygen consumption rate (OCR) is a common proxy for mitochondrial function and can be used to study different disease models. We developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice that is more physiologically relevant than other methods.

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial ...

Neuroscience

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.

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 ...

Biology

Quantitative Analysis of Mitophagy in Murine Pancreatic &#946;-Cells

Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic &#946;-Cells

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in &#946;-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify &#946;-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for &#946;-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in &#946;-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous &#946;-cell models.

Author Spotlight: Assessment of Mitophagy Flux in Pancreatic &#946;-Cells Using Effective and Robust Complementary Approaches 

This protocol outlines two methods for the quantitative analysis of mitophagy in pancreatic &#946;-cells: first, a combination of cell-permeable mitochondria-specific dyes, and second, a genetically encoded mitophagy reporter. These two techniques are complementary and can be deployed based on specific needs, allowing for flexibility and precision in quantitatively addressing mitochondrial quality control.

Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional &#946;-cell mitophagy results in insufficient ...

Analysis of Non-Human Primate Pancreatic Islet Oxygen Consumption

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