Mitochondria, Liver

Clark electrode assays performed for comparative purposes utilized a Hansatech Oxytherm apparatus (PP Systems, Amesbury, MA) for rat heart mitochondria or a Rank system (Rank Brothers, Bottisham, Cambridge, England) for mouse liver mitochondria. For rat heart mitochondria, assays were performed in parallel with the same mitochondrial preparation, MAS, substrates and compounds as for the XF24 assays. Typically 62.5–125 µg of mitochondria were used in a volume of 500 µl MAS plus the appropriate substrate. Respiration was initiated by adding mitochondria, and followed by sequential addition of ADP, oligomycin and FCCP. Concentrations of substrate, ADP, oligomycin, and FCCP were identical to those used in the XF24 experiments. For mouse liver mitochondria, assays were performed in parallel the same mitochondrial preparation, MAS, substrates and compounds as for the XF24 assays with the following modifications: substrate was 5 mM succinate, 2 µM rotenone and 300 µM ADP was used. Typically, 0.3 mg/ml of mitochondria were used in a volume of 2.0–3.5 ml MAS plus the appropriate substrate. Respiration was initiated by adding mitochondria, followed by sequential addition of ADP, oligomycin and FCCP. Concentrations of oligomycin and FCCP were identical to those used in the XF24 experiments. Oxygen consumption rates were converted from nmol O/min/ml to pmol O₂/min/µg mitochondrial protein.

Ethics Statement: Animal housing, euthanasia, and tissue harvest procedures were conducted in accordance with and approved by the UCSD Institutional Animal Care and Use Committee (protocol #S09186) and the Buck Institute Animal Care Committee (protocol #10180). Mitochondria from C57bl/6 (male and female) mice aged 4–6 weeks were isolated by two similar differential centrifugation methods, based upon Schnaitman and Greenawalt [14] (link) or Chappell and Hansford [15] . Specifically, the liver was extracted and minced in ∼10 volumes of MSHE+BSA (4°C), and all subsequent steps of the preparation were performed on ice. The material was rinsed several times to remove blood. The tissue was disrupted using a drill-driven Teflon glass homogenizer with 2–3 strokes. Homogenate was centrifuged at 800 g for 10 min at 4°C. Following centrifugation, fat/lipid was carefully aspirated, and the remaining supernatant was decanted through 2 layers of cheesecloth to a separate tube and centrifuged at 8000 g for 10 min at 4°C. After removal of the light mitochondrial layer, the pellet was resuspended in MSHE+BSA, and the centrifugation was repeated. The final pellet was resuspended in a minimal volume of MSHE+BSA. Total protein (mg/ml) was determined using Bradford Assay reagent (Bio-Rad). Typically, ∼7.5 mg of mitochondria (100 µl volume) was obtained from a single mouse liver. In separate studies in which respiratory rates in the Seahorse and the Rank Clark electrode system were compared, mouse liver mitochondria were isolated according to Chappell and Hansford [15] in 250 mM Sucrose, 5 mM Tris and 2 mM EGTA (STE) on ice. Tissue was homogenized 10 times with a Teflon-glass homogenizer, and the homogenate was centrifuged at 1000 g for 3 minutes (4°C). The supernatant was collected and centrifuged at 11,600 g for 10 minutes. The pellet was resuspended in STE after discarding the whitish layer. The above step was repeated two times to get the final mitochondrial pellet. 8–10 mg of mitochondrial protein was obtained from each mouse liver and resuspended in 400–500 µl of STE.

All experimental procedures were conducted in accordance with EU regulations in the Directive 2010/63/EU of the European Parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes. We have ethical approval from the national Bulgarian Food Safety Agency, Ministry of Agricultural, Food and Forestry (No. 224 from 23 January 2019) and from the local Ethics Committee of Faculty of Biology, Sofia University “St. Kliment Ohridski” (Protocol No. 2/2022). All efforts were made to minimize the number of animals used and their suffering.
The livers for assay of SSAO activity, intact liver mitochondria, and SMPs were isolated from mature male Wistar rats (50–60 days old, supplied by Vivarium with Physiological Laboratory, Project: Creation and Development of Centers of Competence: BG05M2OP001-1.002-0012-C01, “Sustainable utilization of bio-resources and waste of medicinal and aromatic plants for innovative bioactive products”). The animals were housed in cages at 20–24 °C for 12 h light/dark cycle. They received a standard laboratory diet (commercial rat chow, HL-TopMix Ltd., Kaloyanovo, Bulgaria) and water ad libitum and were fasted prior to use. Rats were anesthetized and then decapitated, and the liver was surgically removed.

Young male Wistar rats at 60 days of age and 345.7 g of average body weight were individually caged in a temperature-controlled room and exposed to a daily 12/12 h light/dark cycle with free access to chow diet and drinking water. Rats were divided into two experimental groups according to a different dietary regimen: the first group (control diet, CD) received a standard diet (10.6% fat J/J, 29% protein J/J, 60.4% carbohydrate J/J); the second group (HFD) received high-fat diet (40% fat J/J, 29% protein J/J, 31% carbohydrate J/J). The animals from both groups were fed with the respective diet for 1, 3, 6, 12, or 24 weeks (n = 6 for each group and time point). An additional group (n = 6) was sacrificed at the beginning of the study to establish baseline measurements of body compositions. At the end of the experimental treatments, the rats were anesthetized by i.p. injection of chloral hydrate (40 mg/100 g body weight), decapitated with a guillotine, and the blood was taken from the inferior cava vein. Serum or plasma was obtained by centrifugation at 1000× g for 10 min at 4 °C. The liver and adipose epididymal tissues were quickly excised and transferred in the appropriate buffer to isolate the mitochondria from the liver and the adipocytes from the visceral tissue. All the samples that were not immediately used were stored at −80 °C. Procedures involving animals and their care were conducted in conformity with international and national laws and policies (EU Directive2010/63/EU for animal experiments, ARRIVE guidelines, and the Basel declaration including the 3R concept). The procedures reported here were approved by the Institutional Committee on the Ethics of Animal Experiments (CSV) of the University of Naples Federico II (Permit Number: 2010/0149862) and by the Ministero della Salute.

The recombinant CAC proteins were reconstituted in proteoliposomes as previously described [49 (link)]. Rat liver mitochondria after purification, using the standard procedure of cell disruption and differential centrifugation [50 (link)] were solubilized with 3% Triton X-100 in a PIPES buffer (10 mM) at pH 7.0. Herein, the mitochondrial extract (0.3 mg) were reconstituted in the liposomes as above described for the mitochondrial CAC recombinant protein, in the absence of reducing agents. The concentration of intraliposomal carnitine was 15 mM in all samples.