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

Summary

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.

Abstract

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.

Introduction

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, K_m 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 drawbacks^7,8. 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.

Protocol

1. Cell Culture

1. 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-mercaptoethanol^{9,10,11,12,13}.
2. Remove 832/13 cells from a T75 flask using 2 mL of 0.25% trypsin, incubating at 37 °C for 10 min. Neutralize trypsin by adding 8 mL of complete RPMI 1640 media.
3. Count cells using a hemocytometer and dilute 100 µL of the cell volume from step 1.2 in 900 µL of PBS.
4. Plate 832/13 cells at a density of 2 x 10⁶ cells/mL in a 6 well dish.
5. Culture cells for 48 h in a humidified incubator at 37 °C and 5% CO₂ before beginning respiration experiments.

2. Preparation of Cells for High-resolution Respirometry

1. Treating 832/13 cells with cocoa derived epicatechin monomer.
   1. Culture 832/13 cells for 24 h after plating in a humidified incubator at 37 °C and 5% CO₂.
   2. 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 °C and 5% CO₂.
   3. 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.
      1. 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.
      2. Make 1x Low Glucose SAB with 10 mL of 10x SAB, 100 µL 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.
2. Treating 832/13 cells with curcumin.
   1. Culture 832/13 cells for 48 h after plating in a humidified incubator at 37 °C and 5% CO₂.
   2. 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.
   3. 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 µM curcumin for 30 min. Following incubation, aspirate the buffer.
3. Harvesting cells with trypsin for high-resolution respirometry
   1. After the 3 h incubation in 1x Low Glucose SAB, remove the cells from the plate with 250 µL of 0.25% trypsin per well.
   2. 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.
   3. Count cells using a hemocytometer and dilute 100 µL of the cell volume from step 2.3.2 in 900 µL of PBS.
   4. 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.
4. Harvesting cells with mechanical dissociation for high-resolution respirometry
   1. 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 µL pipette tip.
   2. 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.

3. High-resolution Respirometry

1. Preparation of the high-resolution respirometer.
   1. Turn on the high-resolution respirometer and connect to the desktop by launching the respirometer analysis program.
   2. Aspirate 70% ethanol from both chambers. Soak these chambers in 70% ethanol for a minimum of 45 min.
   3. Wash the chambers twice with 70% ethanol, aspirating after each step.
   4. Wash the chambers three times with ddH₂O, aspirating after each step.
   5. Rinse the chamber plungers in ddH₂O. This cleans off residual ethanol from the plungers and from the titanium port.
2. Calibration of polarographic oxygen sensors.
   1. 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 °C with a data-recording interval at 2.0 s by pushing the F7 button and opening the tab labeled "Systems". 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.
   2. Set the machine at 37 °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 "Oxygen, O₂". 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)).
   3. Following oxygen concentration stabilization, select a region where the change of oxygen concentration is stable to establish background measurement of change in oxygen concentration.
      1. 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 "R1" for each trace, corresponding with each of the two chambers.
      2. Double click on the "O₂ Calibration" box in the bottom left and right corners of the screen, click the "select mark" button for the "air calibration" as R1 and then select "calibrate and copy to clipboard" for both chambers.
3. Evaluation of respiration of 832/13 beta cells prepared in steps 2.1.5 or 2.2.5.
   1. 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 °C for measurement later).
      1. Push plunger in all the way and aspirate the residual volume. Stir the cells continuously throughout the experiment at 750 rpm and 37 °C for all subsequent steps. Make a mark by clicking F4 and labeling as "cells" when samples are loaded.
      2. 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.
   2. After signal stabilization is reached, add 12.5 µL 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 "Glucose" 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.
   3. After signal stabilization is reached add 1 µL of 5mM oligomycin A (2.5 µM final concentration) into each chamber through the loading port. Make a mark measured "OligoA" 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.
   4. After signal stabilization is reached add 1 mM FCCP in 1 µL increments until a maximum respiration rate is established. This represents maximal uncoupled respiration. Between 3-4 µL FCCP is sufficient (1.5-2.0 µM final concentration) to induce maximal uncoupled respiration of INS-1 832/13 cells. Make a mark labeled "FCCP" 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.
   5. After signal stabilization is reached add 1 µL of 5 mM Antimycin A (2.5 µM final concentration) into each chamber through the loading port. Make a mark labeled "AntiA" 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.
4. Measuring 832/13 beta cells for protein concentration and respiration normalization.
   1. Using the 0.5 mL of sample retained at loading, measure protein sample using the BCA method¹³.
   2. Run each sample in triplicate, without dilution, following the manufacturer's instructions.
5. 832/13 beta cell respiration calculations from trypsinized cells.
   1. 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.
   2. Select background readings to normalize the data by selecting and entering into the O₂ Calibration form as described in 3.2.3.
   3. 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.
   4. 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 "copy to clipboard function". This exports the data for use in other analysis programs. Use the "O₂ slope neg." values for calculations.
   5. 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.
6. 832/13 beta cell respiration calculations from mechanically dissociated cells.
   1. 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.
   2. Select background readings to normalize the data by selecting and entering into the O₂ Calibration form as described in 3.2.3.
   3. 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.
   4. 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 "copy to clipboard function". This exports the data for use in other analysis programs. Use the "O₂ slope neg." values for calculations.
   5. 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.

Results

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...

Discussion

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 curcumin^4,5. Treatment with various other compounds cou...

Materials

Name	Company	Catalog Number	Comments
O2k Core: Oxygraph-2k	Oroboros Instruments	10000-02	Instrument for high-resolution respirometry
DatLab 6 Program	Oroboros Instruments	27141-01	Computer program for analysing high-reolution respirometry
INS-1 832/13 cell line	Duke University Medical Center	NONE	Beta cell line, gift from Dr. Christopher Newgard
Curcumin	Sigma	C7727	Pre-treatment of beta cells
Cocoa epicatechin monomer	Virginia Polytechnic Institute and State University	NONE	Pre-treatment of beta cells, gift from Dr. Andrew Neilson
Trypsin	Sigma	T4049	For cell culture
RPMI-1640	Sigma	R8758	832/13 beta cell media
Fetal Bovine Serum	Hyclone	SH30071.03	832/13 beta cell media component
Penicillin-streptomycin	Sigma	P4458	832/13 beta cell media component
HEPES	Sigma	H3662	832/13 beta cell media component/ 1x SAB Buffer
Glutamine	Caisson	GLL01	832/13 beta cell media component
Sodium Pyruvate	Sigma	S8636	832/13 beta cell media component
2-Mercaptoethanol	Sigma	M3148	832/13 beta cell media component
NaCl	Fisher	S271	Component of 10x SAB buffer
KCl	Sigma	P9541	Component of 10x SAB buffer
KH2PO4	Sigma	P5655	Component of 10x SAB buffer
MgSO4	Sigma	M2643	Component of 10x SAB buffer
D-(+)-Glucose Solution	Sigma	G8769	Component of 1x SAB buffer, chemical for respiration assay
CaCl2	Sigma	C5670	Component of 1x SAB buffer
35% BSA Solution	Sigma	A7979	Component of 1x SAB buffer
NaHCO3	Sigma	S5761	Component of 1x SAB buffer
200 proof ethanol	Sigma	459844	Washing Oxygraph O2k Chambers
Oligomycin A	Sigma	O4876	Chemicals for respiration assay
FCCP	Sigma	C2920	Chemicals for respiration assay
Anitmycin A	Sigma	A8674	Chemicals for respiration assay
BCA Protein Kit	Thermo Fisher	23225	For determining protein concentration