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

Deviationp-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 0.993Islets 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.

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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.