*2.1. Animals:*

The study was approved from the Animal Ethics Committee of the University of Duesseldorf, Germany (project identification code: O27/12), and performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The authors ensured that their research complied with the commonly accepted '3Rs': replacement, reduction and refinement.

Male Wistar rats were purchased from the breeding facilities of the University of Düsseldorf (Düsseldorf Germany) or from Janvier Labs (Le Genest-Saint-Isle, France). They were kept at an artificial 12-h light/dark cycle at constant room temperature and humidity with free access to standard chow and tap water.

Forty eight rats (approximately 3 months old) were sacrificed by decapitation under deep sedation with sodium pentobarbital (90 mg/kg) and liver and colon were harvested.

#### *2.2. Preparation of Liver and Colon Homogenates*

Liver and colon homogenates were prepared as described previously [23–25]. Briefly, liver tissue was placed in 4 ◦C cold isolation buffer, minced into 2–3-mm<sup>3</sup> pieces, rinsed twice in isolation buffer to remove traces of blood, and homogenized (Potter-Elvehjem, Pro Scientific, Swedesboro, NJ, USA, 5 strokes, 2000 rpm). Freshly harvested colon tissue was placed in isolation buffer enriched with 2% bovine serum albumin (BSA, Sigma-Aldrich Corporation, St. Louis, MO, USA), longitudinally opened, and dried with a cotton pad to remove remains of faeces and mucus. After incubation with 0.05% trypsin (Thermo Fisher Scientific, Dreieich, Germany) for 5 min, the tissue was transferred into the isolation buffer containing 2% BSA and protease inhibitors (cOmplete™ Protease Inhibitor Cocktail, Roche Life Science, Mannheim, Germany), minced, and finally homogenized (see above). Protein concentration in the tissue homogenates was determined by the Lowry method [26] with bovine serum albumin as a standard. All procedures were performed on ice, all buffer were kept by 4 ◦C.

#### *2.3. Measurement of Mitochondrial Respiratory Rates*

Mitochondrial oxygen consumption was measured as described previously [23–25]. Briefly, the measurement was performed at 30 ◦C using a Clark-type electrode (model 782, Strathkelvin instruments, Glasgow, Scotland). Tissue homogenates were suspended in respiration medium to yield a protein concentration of 4 mg/mL or 6 mg/mL for liver and colon, respectively.

#### 2.3.1. Mitochondrial State 2 Respiration

Mitochondrial state 2 respiration was performed in the presence of either complex I substrates glutamate (Fluka, München, Germany) and malate (Serva Electrophoresis GmbH, Heidelberg, Germany) (both 2.5 mM, G–M) or the complex II substrate succinate (Sigma-Aldrich Corporation, St. Louis, MO, USA) (10 mM for liver, 5 mM for colon, S) combined with 0.5 μM rotenone (Sigma-Aldrich Corporation, St. Louis, MO, USA)—the inhibitor of complex I activity. Rotenon was always added before addition of succinate.

#### 2.3.2. Mitochondrial State 3 Respiration

The maximal coupled mitochondrial respiration (state 3) was measured after the addition of adenosine diphosphate—ADP (Sigma-Aldrich Corporation, St. Louis, MO, USA) (250 μM for liver, 50 μM for colon). The respiratory control index (RCI) was calculated (state 3/state 2) to define the coupling between the electron transport system (ETS) and oxidative phosphorylation (OXPHOS). To reflect the efficacy of OXPHOS, the ADP/O ratio was calculated from the amount of ADP added and oxygen consumed in state 3. The average oxygen consumption was calculated as the mean from three technical replicates.
