Lactate reagent

The glycogen content in cardiac tissue lysate was measured with colorimetric assay kit (BioVision). The PDH activity was determined using a coupled enzyme reaction, which results in a colorimetric product proportional to the enzymatic activity (Sigma). Mitochondria complex I enzyme activity was measured by spectrophotometer as previously reported (Spinazzi et al., 2012 (link)). The lactate level was measured with Lactate Reagent according to manufacturer’s instructions (Trinity Biotech). The insulin level in serum sample was measured using a commercially available kit (Sigma). The blood glucose level was measured by ONE TOUCH Glucose Monitor (Lifescan Inc.) using blood samples obtained by tail bleeding.

Krebs-Ringer-Bicarbonate (KRB) was made containing (98.5 mM NaCl, 4.9 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄7H₂O, 20 mM HEPES, 2.6 mM CaCl-2H₂O, 25.9 mM NaHCO₃). All components were dissolved in deionized H₂O and 5% CO₂ was used to bring KRB to a 7.4 pH. KRB was filtered through a 0.22-µm membrane. Glucose was added to KRB solution and the final concentration was 5 mM. KRB with 5mM of glucose was aliquoted into 48-well plates (Cat# 3548, Corning, NY) and placed into a 37-C cell incubator under 5% CO₂ before dissection and the rest of KRB with 5 mM of glucose was placed on ice in a 35-mm dish for the retina dissections. Neural retinas were dissected from mice and immediately put in KRB with 5 mM of glucose. Single retina was placed in a well of a 48-well plate that was previously placed in the 37-C cell incubator. Lactate released was measured at 30 minutes using the Lactate Reagent (Cat# 73510, Trinity Biotech, Bray, Co Wicklow, Ireland) according to manufacturer protocol and graphed relative to control.

All steps of the assay were performed in Krebs-Ringer-Bicarbonate (KRB).
KRB consisted of 98.5 mM NaCl, 4.9 mM KCl, 1.2 mM KH₂PO₄,
1.2 mM MgSO₄-7H₂O, 20 mM HEPES, 2.6 mM
CaCl-2H₂O, and 25.9 mM NaHCO₃, dissolved in deionized
H₂O, which was bubbled with 5% CO₂ to adjust the pH to
7.4 and filtered through a 0.22 μm membrane. Glucose was diluted with KRB
solution to 5 mM (GKRB), aliquoted into 48 well plates (Cat#3548, Corning) and
placed into a cell incubator at 37°C, 5% CO₂, prior to
dissection. Each retina was dissected and placed in a single well of a 48 well
plate in GKRB. Samples were taken after 15, 30, and 60 minutes and lactate in
the GKRB was measured using Lactate Reagent (Cat#735-10, Trinity Biotech, Bray,
Co Wicklow, Ireland).