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 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 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.
1. Cell Culture
2. Preparation of Cells for High-resolution Respirometry
3. High-resolution Respirometry
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, nonphosphorylating respiratory state. Maximal respiration will be achieved when the beta cells are treated with the uncoupling agent FCCP, which is defined as ETS respiration, or electron transport system respiration. Finally, respiration will drop to zero when treated with Antimycin A, labeled as ROX (residual oxygen consumption) which refers to the non-respiratory cellular oxygen consumption.
Differences in cell number will significantly change the quality of the data obtained from this technique. Glucose-induce respiration was measured at 0.5, 1, and 2 x 106 cells/mL (Figure 2A-2C). Using cell counts and BCA protein assays, protein concentrations corresponding to the three tested cell concentrations were calculated (Figure 2D). Measurements using 0.5 x 106 cells/mL failed to demonstrate glucose-stimulated respiration (Figure 2A), and only the ETS associated increased respiration was shown to be significant. This suggests that the low cell number results in a low signal to noise ratio that confounds the results. Similarly, measurements at 2 x 106 cells/mL also resulted in significant variability, as indicated by statistical significance only being reached for the ETS measurements (Figure 2C). This is presumably due to the need to re-oxygenate the chambers multiple times during the assay, which resulted in greater variability from sample to sample. These data demonstrate that concentrations of 1 x 106 cells/mL result in significant glucose-stimulated respiration, LEAK and ETS measurements, which are necessary for respiration analyses (Figure 2B and 2E).
Two methods for removing the cells for respiration are presented in this protocol. Using trypsin to remove cells is effective when measurements are completed at 37 °C. Respiration measurements done in the absence or presence of curcumin (Figure 3A) demonstrates maintenance of glucose-stimulated respiration, LEAK and ETS respiration. Comparison of curcumin treated cells to untreated cells demonstrates no change to any of these respiratory parameters (Figure 3B-C). Respiration measurements done in the absence or presence of cocoa epicatechin monomer (Figure 3D) also demonstrates maintenance of glucose-stimulated respiration, LEAK and ETS respiration. Comparison of cocoa epicatechin monomer treated cells to untreated cells demonstrates increased respiration at 2.5 mM and 16.7 mM Glucose, as well as in LEAK and ETS respiration (Figure 3E-F). These data demonstrate that while curcumin does not enhance 832/13 beta cell respiration, cocoa epicatechin monomer does (Figure 3G). Curcumin has been shown to enhance insulin secretion through inhibiting phosphodiesterase activity14. Our data suggest that curcumin does not enhance insulin secretion through modulating mitochondrial function. We have already demonstrated that culture in the presence of cocoa epicatechin monomers induces expression of various components of the mitochondrial electron transport chain5. We also observed increased respiration in the LEAK state (induced by treatment with oligomycin) and with ETS (electron transport system, induced by FCCP). The use of this tool suggests a number of potential targets for future studies.
As an alternative to trypsin removal of cells, mechanical dissociation may also be performed using the presented washing technique when completed at 37 °C. Respiration measurements were done in the absence or presence of curcumin or cocoa epicatechin monomer (Figure 4A-C). These data demonstrate that the trends observed with trypsin preparation of the cells are maintained, however the sensitivity appears to be diminished. While control and cocoa epicatechin monomer treated cells maintained the glucose-stimulated, LEAK and ETS respiration, cells treated with curcumin only retained the glucose-stimulated respiration due to the increased sample-to-sample variability. While the trends observed with trypsin cell preparations were maintained (Figure 4D-F), the significance decreased in all parameters, presumably to the greater sample-to-sample variability introduced by the mechanical dissociation and the normalization to protein concentration rather than cell number.
These data demonstrate that this protocol can be used to measure respiration of intact INS-1 832/13 beta cells. Furthermore, our protocol suggests an optimum cell number for accurate measurements and a preferred method for preparing the cells to maximize the signal to noise ratio.
Figure 1: Representative respiration curve of intact 832/13 beta cells. Intact 832/13 beta cells are measured for respiration in (Figure 1A) 2.5 mM Glucose, 16.7 mM Glucose, 2.5 µM Oligomycin, 2 µM FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) and 2.5 µM Antimycin A. An expansion of the 16.7 mM Glucose section (Figure 1B) is presented to demonstrate the increase in glucose-stimulated respiration. Cells were measured at a concentration of 1 x 106 cells/mL. LEAK refers to the basal, nonphosphorylating respiratory state, ETS refers to the uncoupled respiratory capacity of the electron transport system, and ROX refers to the residual oxygen consumption after mitochondrial respiration is inhibited. Please click here to view a larger version of this figure.
Figure 2: Determination of number of 832/13 beta cells necessary for glucose-induced respiration measurements. Intact 832/13 beta cells are measured for respiration at concentrations of (A) 0.5 x 106 cells/mL (B) 1 x 106 cells/mL, or (C) 2 x 106 cells/mL. (D) Protein concentrations that correspond with 0.5 x 106 cells/mL, 1 x 106 cells/mL, and 2 x 106 cells/mL. (E) Comparison of respiration at 0.5 x 106 cells/mL, 1 x 106 cells/mL, and 2 x 106 cells/mL. Respiration was measured under 2.5 mM Glucose, 16.7 mM Glucose, 2.5 µM Oligomycin, 2 µM FCCP and 2.5 µM Antimycin A conditions. Respiration with 2.5 and 16.7 mM glucose demonstrate the glucose-stimulated respiration response of the beta cells. Respiration after oligomycin demonstrates the LEAK respiration (basal, nonphosphorylating respiratory state). Respiration after FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) treatment demonstrates (uncoupled respiratory capacity of the electron transport system, ETS). n = 6 independent runs. Data is presented as average ± SEM. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. Please click here to view a larger version of this figure.
Figure 3: Respiration measurements of 832/13 beta cells after release using trypsin. 832/13 beta cells were cultured in the presence or absence of monomeric cocoa epicatechin or curcumin and respiration was measured after releasing cells using trypsin. Respiration was measured under 2.5 mM Glucose, 16.7 mM Glucose, 2.5 µM Oligomycin, 2 µM FCCP and 2.5 µM Antimycin A conditions. Respiration with 2.5 and 16.7mM glucose demonstrate glucose-stimulated respiration. Respiration after oligomycin demonstrates the LEAK respiration (basal, nonphosphorylating respiratory state). Respiration after FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) treatment demonstrates (uncoupled respiratory capacity of the electron transport system, ETS). Respiration after antimycin A treatment demonstrates residual oxygen consumption (ROX). (A) Treatment of 832/13 cells with curcumin maintains the appropriate changes in respiration due to the treatments. (B) Representative trace and (C) average of data for cells treated with curcumin demonstrates no change to 832/13 beta cell respiration. (D) Treatment of 832/13 cells with cocoa epicatechin monomer maintains the appropriate changes in respiration due to the treatments. (E) Representative trace and (F) average of data for cocoa epicatechin monomer treated cells demonstrates increased respiration in all parameters after cocoa epicatechin monomer treatment. (G) All three parameters are presented together for comparison. n = 6 independent runs. Data is presented as average ± SEM. *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001. Please click here to view a larger version of this figure.
Figure 4: Respiration measurements of 832/13 beta cells after release using mechanical dissociation. 832/13 beta cells were cultured in the presence or absence of monomeric cocoa epicatechin or curcumin and respiration was measured after releasing cells using mechanical dissociation. Respiration was measured under 2.5 mM Glucose, 16.7 mM Glucose, 2.5 µM Oligomycin, 2 µM FCCP and 2.5 µM Antimycin A conditions. Respiration with 2.5 and 16.7 mM glucose demonstrate the glucose response of the beta cells. Respiration after oligomycin demonstrates the LEAK respiration (basal, nonphosphorylating respiratory state). Respiration after FCCP (Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) treatment demonstrates (uncoupled respiratory capacity of the electron transport system, ETS). Average of data for cells (A) untreated cells or cells treated with (B) curcumin or (C) cocoa epicatechin monomer demonstrate that mechanical dissociation maintains glucose-stimulated respiration in all treatments, while LEAK and ETS significance are lost only in curcumin treated cells. Comparison of untreated cells to (D) curcumin or (E) cocoa epicatechin monomer demonstrates increased respiration in all parameters with cocoa epicatechin monomer with no significant change to respiration with curcumin. (F) All three parameters are presented together for comparison. n = 6 independent runs. Data is presented as average ± SEM. *p <0.05, **p <0.01, ****p <0.0001. Please click here to view a larger version of this figure.
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 could be used in this protocol, with pretreatment (as shown here) or by treating within the respiratory chambers with minimal modifications to the base protocol.
There are various ways by which this method could be modified. Other beta cell lines could be used; however, the cell number and SAB wash times will need to be determined experimentally. Primary rodent and human islets could also be used; however, the number of cells or islet equivalents will also need to be determined experimentally. At least one study has measured islet oxygen consumption using high-resolution respirometry15. This study required 400 islets per reaction, but did not measure glucose induced respiration changes. Given that primary tissue is frequently used with high-resolution respirometers16,17, intact islets could be used after experimental troubleshooting. However, given the large number of islets required for this assay, other methods of measuring oxygen consumption may be better suited for measurements of primary islet respiration.
There are a few critical steps with the presented protocol. First, having an adequate number of cells is critical. Our studies show that 832/13 beta cells at a concentration of 1 x 106 cells/mL is sufficient for glucose-stimulated respiration measurements. Measurements using 0.5 x 106 cells/mL demonstrated low glucose-stimulated respiration and a low signal to noise ratio. Furthermore, measurements using 2 x 106 cells/mL resulted in high sample to sample variability, and rapid cellular use of oxygen which required chamber re-oxygenate and frequently resulted in rapid cell death. The appropriate number of cells should be determined experimentally for the given cell line in order to troubleshoot this critical step.
A second critical step is preparing the beta cells to respond to elevated glucose levels. The RPMI 1640 culture media has high glucose levels. The cells must be moved from the high glucose to a low glucose culture. A 3 h incubation in 1x Low Glucose SAB is sufficient for the beta cells to have a significant response to the addition of ~16.7 mM glucose. Wash periods of less than 3 h result in variable responses to the addition of glucose. Troubleshooting glucose response problems can be addressed by increasing the length of SAB wash time.
A third critical step is the method for preparing the cells for measurements. Our data demonstrates that while trypsin and mechanical dissociation can both be used, trypsin released cells result in more consistent readings and less sample-to-sample variability. The trend observed with both methods was maintained, however the strength of the data was greater with the cells prepared using trypsin. While other methods, such as cell scraping, may be used our data suggest that the use of trypsin will result in the most reproducible results with less sample-to-sample variability18,19.
Limitations of this approach include the following. First, with only two treatment chambers, this is not a high throughput screening method. Each run of two samples takes between 3-4 h, including the time to prepare the machine. Second, a relatively large concentration of cells is required for the assay, given the ~2.2 mL of sample (or ~2.2 x 106 cells) needed per chamber. Due to the amount of sample required for the assay, the use of this tool and protocol for primary islets is limited unless a large supply is available. The previous studies that have used islets for respiration in this technique used ~400 islets/chamber15. This would require, roughly, islets from two mice for each chamber20. Third because permeabilized cells are not being used, the ability to determine the effect of the treatment on individual electron chain components by using compounds that are not cell permeable is not available.
There are a number of significant advantages of this protocol with respect to existing methods. First, this protocol allows for the stepwise titration of various membrane permeable compounds. This allow for the accurate determination of necessary concentrations. Second, use of intact beta cells allows us to query the effect on cytosolic and mitochondrial glucose metabolism7. This allows us to observe phenotypes indicative of changes to the glycolytic pathway, an observation that is lost when using permeabilized cells. Third, the intact cells allow us to use the response to elevated glucose as a metric of beta cell function, which is lost in permeabilized variants of this assay. Finally, this protocol allows for the addition of various and multiple compounds to query the effect of compounds on beta cell respiration, a luxury that is not present with other tools that measure respiration.
Future applications of this method include the ability to measure parameters such as ATP, calcium, and H2O2 levels concurrent with respiration measurements. This method also allows for measurement of respiration in the presence of other fuel sources, such as fatty acids or ketones. Finally, with some modifications, measuring secreted insulin levels concomitant with respiration measurements may also be possible.
The authors have nothing to disclose.
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.
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 |