A Novel High-Throughput Assay for Islet Respiration Reveals Uncoupling of Rodent and Human Islets

Abstract

BackgroundThe pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR) may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.Methodology/Principal FindingsThe XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.Conclusions/SignificanceThe assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

Rpl13a small nucleolar RNAs regulate systemic glucose metabolism

“…While markers of inflammation and β cell replication were indistinguishable between the genotypes (Supplemental Figure 5), streptozotocin-induced accumulation of the oxidatively modified lipids 4-hydroxynonenal and 3β,5α,6β-cholestanetriol (triol) was prevented in Rpl13a-snoless mice ( Figure 7, C and D). Furthermore, TUNEL staining of insulinpositive β cells was decreased by 50% in Rpl13a-snoless animals proton leak relative to ATP production in both genotypes ( Figure 5A), similar to results previously described in WT islets (15). At 20 mM glucose, basal and maximal respiration were substantially increased in Rpl13a-snoless islets relative to WT, which was due to a large increase in proton leak, but oxygen consumption for ATP production was not significantly different ( Figure 5B).…”

“…Oxygen consumption of islets was measured using a Seahorse XF24 analyzer (Seahorse Bioscience) (15). Fifty islets were hand picked and washed once in assay media containing 1% FBS and 1 mM l-glutamine, and 3 mM or 20 mM glucose, and then transferred into the depressed well of an XF24 Islet Capture Microplate.…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Indeed, apart from the general consensus that ␤-cell mitochondria possess a high endogenous proton leak (2,4,542), we lack any information on the extent to which they have evolved distinctive properties integral to the physiological function of the ␤-cell.…”

“…These include the abnormally high endogenous mitochondrial proton leak (2,542) and the largely unexplored quantitation of ATP utilizing pathways (but see Refs. 178,502).…”

- Consumption of Fructose-Sweetened Beverages for 10 Weeks Reduces Net Fat Oxidation and Energy Expenditure in Overweight/Obese Men and Women

“…Oligomycin addition was followed by the addition of carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), an uncoupler of electron transport that induces peak oxygen consumption (an indirect indicator of peak oxidative metabolism) at final concentration 1.25 μM. Rotenone was then added in 1.0 μM final concentration to reveal nonmitochondrial respiration and end the metabolic reactions [33,34]. Extracellular acidification, an indirect measure of glycolytic capacity, and oxygen consumption, a measure of oxidative metabolism was measured using the SeaHorse XF24 Extracellular Analyzer from SeaHorse Bioscience (Billerica, MA).…”