TITLE: U Mass - Organ-specific glucose uptake
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>Glucose uptake in individual organs can be measured using a bolus injection of 2-deoxy-D-[1-</span><span style = "vertical-align:super;">14</span><span>C] glucose, a non-metabolizable glucose analog, and by determining labeled metabolite levels in select tissues. Insulin resistance is characterized by reduced glucose metabolism and develops in obese mice.</span></div></div>

[STEPS]
?. Survival surgery is performed to establish a chronic indwelling catheter at 5~6 days prior to experiment for intravenous infusion.  (refer to M1023: Surgery-jugular vein cannulation)
?. Mice are fasted overnight (~15 hours) or for 5 hours prior to the start of experiment.
?. Place a mouse in a rat-size restrainer with its tail tape-tethered at one end.
?. Administer an intravenous bolus injection of 10 Ci of 2-deoxy-D-[1-14C] glucose (2-[14C]DG) in awake mice. Alternatively, intraperitoneal injection of 10 Ci of 2-[14C]DG may be used in awake mice.
?. After 30 min, rapidly freeze-clamp the tissues in liquid N², and store tissue samples in -80ºC freezer for biochemical assay.
?. Biochemical assay is conducted using frozen tissue samples (e.g., skeletal muscle, adipose tissue, heart) to measure tissue levels of 2-[14C]DG-6-phosphate.a) Prepare a heat block set to ~ 100°C.b) Prepare anion-exchange columns by washing with 5 ml of dH²O. c) Homogenize 50–100 mg of frozen tissue samples by adding ten times the volume of dH²O (50 mg of tissue in 500 µl of dH²O) in glass tubes using a tissue homogenizer. d) Following homogenization, place the glass tubes in the heat block for 10 min, vortex for 2 sec, and then cool to room temperature. e) Transfer the homogenized samples to microcentrifuge tubes using transfer pipettes and centrifuge at 16,000 × g for 5 min. f) Add 33 µl of homogenate (supernatant) to 467 µl dH²O in a scintillation vial labeled “total” sample. g) Add 5 ml of scintillation cocktail, vortex, and count the samples for 14 C using a liquid scintillation counter (total 14C samples). h) Transfer 333 µl of homogenate (supernatant) to the anionexchange columns for the separation of 2-[14C]DG-6-P from 2-[14C] DG. i) Wash the columns with 2 ml of dH²O three times and collect the samples into a scintillation vial labeled “wash” sample. j) Vortex the “wash” samples, and transfer 500 µl of “wash” samples to another set of scintillation vials to be counted for 14C using a liquid scintillation counter (wash samples containing 2-[14C ] DG). k) Elute the columns with 2 ml of 0.2 M formic acid/0.5 M ammonium acetate three times, and collect the samples into a scintillation vial labeled “eluate” sample. l) Vortex the “eluate” samples, and transfer 500 µl of “eluate” samples to another set of scintillation vials to be counted for 14C using a liquid scintillation counter (eluate samples containing 2-[14C] DG-6-P).
?. The rate of glucose uptake in individual organs is determined using 2-[14C] DG. 2-[14C] DG is taken up by cells, phosphorylated by glucokinase to become 2-[14C] DG-6-P, and not further metabolized. Thus, organ-specific accumulation and level of 2-[14C] DG-6-P following a bolus injection of 2-[14C] DG reflect glucose uptake in individual organs.