Mitochondrial respiration assay We employed high-resolution respirometry with a previously published selleckchem substrate-inhibitor protocol that permitted rapid analysis of ETS function and integrity in tissue homogenates as well as permeabilised cardiac fibres.22,23 We elected to use tissue homogenates because these provided a means for rapid simultaneous processing of the several tissues for measurement of tissue-specific mitochondrial respirational flux rate. Homogenates were also considered superior to mitochondrial isolation in this experimental setting as they sum the entire mitochondrial population present in tissue samples. This approach avoids the processing delays of enriched organelle preparations as well as the risk of any potential confounding bias that can result if swollen, more fragile and/or damaged mitochondrial subpopulations are lost through the processing steps required for pure mitochondrial isolation.

23�C25 The tissues studied were: the pancreas (tail and head separately), duodenum, mid-jejunum, lung (left lower lobe), heart (left ventricular endomyocardium), left kidney and liver (left lobe). All tissues with the exception of the left ventricular endomyocardium, were cut into small pieces (~2 mm2), quickly blotted dry, weighed and placed into ice-cold respiration assay media [0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionate, 20 mM taurine, 10 mM KH2PO4, 110 mM sucrose and 1 mg/ml bovine serum albumin (BSA) in 20 mM HEPES, pH 7.1 at 30��C] containing one Complete? protease inhibitor tablet (Roche, Basel, Switzerland) per 30 ml and 20 mg/ml fatty acid free BSA.

These tissues were homogenised immediately before assay. The permeabilised left ventricular endomyocardial fibers were prepared according to a published methodology.22 Approximately 25 mg was placed into a droplet of ice-cold high-energy relaxing solution [10 mM EGTA-Ca2EGTA buffer (free Ca2+ concentration 0.1 ��M), 9.5 mM MgCl2, 3 mM KH2PO4, 20 mM taurine, 5 mM ATP, 15 mM creatine phosphate, 49 mM K+ MES, 29 mM imidazole-HCl, pH 7.1] and dissected into fibre bundles of ~0.5 �� 1 mm. The dissected fibre bundles were transferred into 1 ml of fresh high-energy relaxing solution plus 50 ��g saponin and gently stirred for 30 min at 4��C for permeabilisation. They were then washed three times in ice-cold respiration medium to remove the saponin and adenine nucleotides.

Fibre bundles were then weighed after removing adherent liquid by blotting on lint-free lens tissue. Mitochondrial respiration was measured in parallel 2-ml chambers using an OROBOROS? Oxygraph 2 K (Anton Paar, Graz, Austria). The respiratory measurements were performed at 30��C and the oxygen concentration at air saturation of the medium was 215 nmol O2 per Carfilzomib ml at 95 kPa barometric pressure.26 For pancreatic and intestinal tissues, an additional 10 mg/ml of fatty acid-free BSA was added to the respiration medium.