The study compared measurements of oxygen consumption rate (OCR) in pancreatic islets from pigs and non-human primates using an extracellular flux analyzer and a stirred microchamber method. The analyzer produced comparable OCR measurements to the standard method but with higher variability. Basal OCR was lower in non-human primate islets than pig islets, but primate islets showed a greater increase in OCR in response to hyperglycemia. Immature pig islets had almost no OCR response to glucose. The analyzer can provide species-specific and maturation state insights into islet function and mitochondrial health beyond what the standard method allows.

1. Measurement of oxygen consumption rate of porcine and non-human primate pancreatic islets using
an extracellular flux analyzer compares closely with stirred microchamber method and provides
additional functional insights
Joshua J. Wilhelm1, Zachary A. Swanson1, Kate R. Mueller1, and Bernhard J. Hering1
1Schulze Diabetes Institute, Department of Surgery, University of Minnesota, Minneapolis, MN
Conclusions
There was no statistical difference between OCR measurements of the two
methods for all experiments (n=6); however, the standard deviations for the flux
analyzer were 2-3 fold higher than the stirred microchamber method and the
average measured OCR/DNA value was 10% lower with the XF analyzer. In
both assays, baseline OCR/DNA was much lower for NHP-Rhesus (n=3) than all
porcine islets (n=4), consistent with that seen historically. In both species, we
observed a trend where islets with a greater basal OCR were able to respond
more significantly to hyperglycemia - average ΔOCRmax for NHP-Rhesus islets
after glucose injection was 6-fold greater than low-yield porcine islets (n=2), and
20-fold greater than functionally immature porcine-neonatal islets (n=1).
Interestingly, ΔOCRmax after glucose injection for the genetically distinct high-
yield porcine islets (57%) was slightly greater even than NHP-Rhesus islets
(52%).
Experimental Design
Background
Islets from four porcine (3 adult, 1 neonatal) and two non-human primate (NHP)
isolations cultured for 6-14 days were used in this study. Islets were seeded
using a specially adapted islet capture microplate. On the day of the experiment
culture media was removed and XF assay media +5.6 mM glucose was used to
wash islets. 50-100 islets were seeded by concentration and allowed to
equilibrate for 30 minutes at 37°C, 0% CO2.. OCR was measured at 5.6 mM
glucose every 8 minutes for 5 timepoints and baseline OCR was used from
timepoint 3 to allow for stabilization of measurements. Glucose concentration
was increased to 15.6 mM by injecting 56 uL of 156 mM glucose and 6 more
measurements were taken every 8 minutes thereafter. OCR values for each
method were compared and analyzed using an unpaired student t-test (flux
analyzer n=20, stirred microchamber n=3). The ΔOCRmax after glucose injection
was calculated as the difference between the baseline (injection point) and the
highest peak after injection.
Results
Oxygen consumption rate (OCR) is a measure of mitochondrial function and
has been shown by our group previously to correlate with success of animal and
human clinical islet transplantation. The rate of oxygen consumption provides
insight into how well the oxidative phosphorylation pathway is coupled to energy
production in the form of ATP. During acute hyperglycemic instances, greater
mitochondrial function allows islets to efficiently supply enough ATP to trigger
insulin release, thus returning the body to normoglycemia. Our current gold
standard assay for measuring OCR of islets is the stirred microchamber (SM)
method, which is performed in a closed chamber, but is limited to monitoring
changes in pO2 and requires a large sample. We sought to determine whether
the use of an extracellular flux (XF) analyzer (Seahorse XF24, Seahorse
Biosciences) is comparable to the SM method. The XF analyzer requires fewer
islets (2.5-10x less), and can monitor samples dynamically and in real-time,
while adding compounds through 4 injection ports in each sample well and
monitoring the effects. In addition to OCR, the XF analyzer simultaneously
measures extracellular acidification rate (ECAR), a measure of ATP production
through glycolysis, and which can be used alongside OCR to yield a
bioenergetic phenotype. While determining the comparability of these two
methods, we sought to utilize the additional capabilities of the XF analyzer to
explore novel functional outputs that might help explain differences in islet
products based on species, maturation state, or breed of the donor from which
the islets were derived.
The XF analyzer can be used to obtain OCR measurements comparable to the
SM method. The high standard deviations could possibly be due to assay media
composition, heterogeneity of the small islet sample in the individual wells (# of
beta cells or function of subpopulations), or further technical refinements that have
yet to be elucidated. In addition to the method comparability of baseline OCR, we
showed with the XF analyzer that there are clear species differences between
islets in responding to acute hyperglycemia using ΔOCRmax. Basal NHP OCRs
were nearly half the basal OCR of the porcine preparations, yet the NHP islets
were more responsive to hyperglycemia. This supports the general notion that a
higher basal OCR may indicate health or resiliency, but species must be taken into
account. Functional immaturity of neonatal pancreas tissue is supported by the
observation that there was almost no ΔOCRmax upon stimulation, even with
comparable basal OCR/DNA. Similar species and maturation-specific differences
in insulin secretion were previously reported by our group. The XF analyzer
therefore potentially offers the opportunity to monitor maturation of neonatal islet
cultures ex vivo and examine species differences in coupling efficiency, although
more data is needed to confirm these initial findings. Further functional
characterization can be explored with the XF analyzer using the injection ports to
deliver drugs that manipulate the aerobic and anaerobic respiratory machinery of
the islets, to explore the mitochondrial quality, durability and bioenergetic
phenotype of the islets. Similar methods are currently being used to study and
phenotype cell dysfunction in disease models such as cancer, chronic heart
failure, cirrhosis, and type 2 diabetes.
Experiment ID
Stirred-
Microchamber
OCR
Standard
Deviation
Seahorse
OCR
Standard
Deviation
p-value
NHP-Rhesus 119.5 17.67 108.69 36.26 0.6623
Porcine-Low Yield 216.4 21.13 215.19 35.86 0.9557
Porcine-Low Yield 240.7 26.3 234.48 76.47 0.8921
Porcine-High Yield 259.3 9.7 185.73 74.01 0.1065
NHP-Rhesus 145.5 8.14 114.92 43.1 0.2425
Porcine-Neonatal 158 9.01 158.23 44.2 0.993
Islets in a screen covered well Islet plate view
Basic use of XF technology (above) uses oxygen
and pH sensors to measure changes in well. Phenotype
profile (Right) of an energetic islet assay.
Figure 1- A. Typical OCR graph normalized to DNA. Islets introduced to 15mM glucose at point A show increased OCR
after injection. B. Typical OCR graph after normalized to baseline %. Islets introduced to 15.6mM glucose increase
significantly while OCR of islets in constant glucose remains steady.
1A 1B
Future Work
References
A mitochondrial stress test can be done using a series of drug injections that each
modifies a particular point in the oxidative phosphorylation pathway. Simultaneous
monitoring of the changing OCR following each injection yields a mitochondrial
profile that includes basal OCR, coupling efficiency, non-mitochondrial respiration,
and the spare respiratory capacity of the islets. We hope to use this information to
elucidate aspects related to species differences as well as overall health of an islet
preparation.
Hill, B. G., Benavides, G. A., Lancaster, J. R., Ballinger, S., Dell’Italia, L., Zhang, J., & Darley-Usmar, V. M. (2012). Integration of cellular
bioenergetics with mitochondrial quality control and autophagy. Biological Chemistry,393(12), 1485–1512. doi:10.1515/hsz-2012-0198
Papas, K.K., Pisania, A., Wu, H., Weir, G.C., and Colton, C.K. (2007). A Stirred Microchamber for Oxygen Consumption Rate Measurements
With Pancreatic Islets. Biotechnol. Bioeng. 98, 1071–1082.
Papas, K.K., Colton, C.K., Nelson, R.A., Rozak, P.R., Avgoustiniatos, E.S., Scott, W.E., Wildey, G.M., Pisania, A., Weir, G.C., and Hering, B.J.
(2007b). Human Islet Oxygen Consumption Rate and DNA Measurements Predict Diabetes Reversal in Nude Mice. Am. J. Transplant.
Off. J. Am. Soc. Transplant. Am. Soc. Transpl. Surg. 7, 707–713.
Wikstrom, J. D., Sereda, S. B., Stiles, L., Elorza, A., Allister, E. M., Neilson, A., … Shirihai, O. S. (2012). A Novel High-Throughput Assay
for Islet Respiration Reveals Uncoupling of Rodent and Human Islets. PLoS ONE, 7(5), e33023. doi:10.1371/journal.pone.0033023
Figure 2- NHP Rhesus islets
demonstrate lower basal
OCR values than porcine
islets, but are observed to
be much more responsive
when stimulated with
glucose. Porcine neonatal
islets had almost no
response to glucose
stimulation as expected due
to their maturation state,
which has limited beta cell
mass. Interestingly, genetic
background of porcine
donors seems to play a role
in ΔOCRmax as a genetically
distinct high-yield donor had
ΔOCRmax values similar to
NHP-Rhesus donors, while
low-yield donors of another
breed did not.