U 13c glucose

Fatty acid labeling was performed as published previously (Tumanov et al., 2015 (link)). DMEM supplemented with 10mM U-13C-glucose and 2mM U-13C-glutamine (sigma) and 10% dFBS was used as indicated. After 72h incubation, cells were placed on ice and medium was aspirated, The cells were then washed 2x with ice-cold PBS, quenched with 0.75 mL of 1:1 v/v PBS:Methanol kept at −20°C. After cell scraping, the extraction solvent was transferred to glass tubes, and the total fatty acids was extracted in 0.5 mL chloroform (kept −20°C) and dried under nitrogen gas. Lipids were saponified and methylated with toluene, methanol and methanolic-HCL, and then vortexed and incubated at 100°C for 60 min. Fatty acid methyl esters were extracted with water and hexane and hexane fraction analyzed by GC/MS (Tumanov et al., 2015). Fraction de novo synthesized equals 1-fraction M0.

Isolated mouse retina or confluent human fetal RPE cells were changed into pre-warmed Krebs-Ringer bicarbonate buffer (KRB) containing, depending on the experiment, [1,2] ¹³C glucose, U-¹³C glucose, or U-¹³C lactate (Sigma) as described elsewhere (Du et al., 2013a (link); Du et al., 2015 (link); Du et al., 2016b (link)). Both retinas and RPE cells were incubated for the specified time points. Metabolites from each time point were extracted and analyzed by gas chromatography mass spectrometry (GC-MS, Agilent 7890/5975C) as described in detail (Du et al., 2013a (link); Du et al., 2013b (link)).

C57BL/6J mice were raised at the animal facility at the University of Ulm under specific pathogen-free (SPF) conditions, receiving a standard laboratory diet and water ad libitum. Fresh faecal pellets were randomly collected from 12 healthy animals within 4 h of excretion. The pellets of all animals were finally pooled and subsequently homogenized in M9 minimal medium [34 (link)] supplemented with thiamin (2 mg/L) and Casamino Acids (1 g/L), lacking glucose at this stage, to obtain a 15% (w/v) faecal slurry. Slurry material (1 mL for each treatment) was mixed 1 : 1 with M9 minimal medium containing 80 mM of [U¹³C]glucose (≥99 atom%) (Sigma-Aldrich GmbH, Steinheim, Germany), yielding a final concentration of 40 mM glucose and 7.5% (w/v) of faecal material. Samples were incubated in individual 15 mL reaction tubes at 37°C for 0 h (control), 2 h, or 4 h under anaerobic conditions, which were obtained by using airtight jars and AnaeroGen sachets (Merck, Darmstadt, Germany) resulting in a total of three different incubation treatments. Prior to use, the M9 minimal medium was filter sterilized, deoxygenated, and prewarmed to 37°C.

Mice were fasted for 6 hours prior to all blood sampling. For glucose absorption analysis 10μl/g mouse of U¹³C-glucose (0.5 M, Sigma) or vehicle (saline) was gavaged and allowed to be absorbed for 15 min. Mice were then anesthetized and the portal vein blood was sampled by cutting the portal vein with small scissors. For re-feeding experiments mice a commercially available whey-based protein supplement (QNT) suspended in saline was gavaged at 1.56 g/kg mouse and allowed to be absorbed for 30 min. Mice were then anesthetized and systemic blood was sampled from the heart. For both experiments serum was frozen until analysis.