D 2 3h glucose

2% Isoflurane, 8-0 polyprolene suture, buprenorphine 0.01 mg/kg, 2% Evan’s Blue, 1% 2, 3, 5-triphenyltetrazole (TTC, Sigma, T8877-100g), Leica microscope (Germany), biochemical analyzer, d-glucose 7 mM, insulin 10 mU/L, Oleate 0.4 mM, 1% bovine serum albumin (BSA), [9, 10]-³H-oleic acid (50 mci/L), ¹⁴C-glucose (20 mci/L), d-[2–3H] glucose (1 mci/L) and d-[5–3H] glucose (1 mci/L) were purchased from Perkin Elmer Company in the United States.

Each counterflow assay was performed by adding 2 μL proteoliposomes into 98 μL KPM buffer containing 1 μCi (0.427 μM) D-[2-³H]-glucose (PerkinElmer). After incubation for 30 s, solution was aspirated through 0.22 μm GSTF filter (Millipore). Then the filter was rinsed with 2 mL ice-cold KPM buffer and incubated with 0.5 mL Optiphase HISAFE 3 (PerkinElmer) overnight for liquid scintillation counting.
For the measurement of K_m and V_max, unlabeled glucose was added into KPM buffer at the indicated concentration, and the initial velocities were measured in 15 s. For the measurement of IC₅₀ value, proteoliposomes were preincubated with the indicated concentrations of CCB (Sigma) for 30 min before adding into reaction buffer. All experiments were repeated for three times and data were examined by GraphPad Prism. Error bars represent SD.

All counterflow assays were performed at 25 °C. For each assay, an aliquot of 2 μl proteoliposomes was added into 98 μl KPM 6.5 buffer plus 1 μCi D-[2-³H] glucose (specific radioactivity 23.4 Ci/mmol, PerkinElmer) or D-[³H] xylose (20 Ci/mmol, American Radiolabeled Chemicals, Inc.). The final concentrations of the external D-[2-³H] glucose and D-[³H] xylose were 0.42 μM and 0.5 μM, respectively. Uptake of radio-labeled substrates was terminated at 30 s by rapidly filtering the solution through 0.22 μm GSTF filters (Millipore) and washed with 2.5 ml KPM 6.5 buffer. The filter was solubilized with 0.5 ml Optiphase HISAFE 3 (PerkinElmer) for 30 min for liquid scintillation counting with MicroBeta JET (PerkinElmer). Liposome without protein was used as negative control.
For inhibition assays, an aliquot of 2 μL proteoliposome pre-incubated with 100 μM indicated inhibitors for 0.5 h on ice was added to 98 μL KPM buffer containing 1 μCi D-[2-³H]-glucose or D-[³H] xylose with 100 μM inhibitors. To determine the IC50 of the inhibitor, a series of concentrations of the inhibitor was applied as indicated.
All experiments were repeated at least three times. Data for liposome-based counterflow assay were analyzed using GraphPad Prism 6.