TITLE: Vandy - Hyperinsulinemic-euglycemic Clamp
AUTHORS: Li Kang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Mice with catheters implanted in the jugular vein (infusions) and carotid artery (sampling) are used for this procedure (V3002). The hyperinsulinemic, euglycemic clamp or “insulin clamp” has been used in a variety of species to assess insulin action. In an insulin clamp, the rate of glucose infused to maintain euglycemia is an index of whole body insulin sensitivity. Isotopes can be used during an insulin clamp to distinguish between insulin’s effects on endogenous glucose production and glucose utilization, or to examine insulin’s effects on specific tissues and metabolic pathways. The insulin infusion rate used in a study depends on a number of factors such as whether the experiment requires a sensitive analysis of endogenous glucose production, or whether the mouse is known to be unusually insulin resistant (e.g. ob/ob).  Finally the insulin clamp technique, especially when isotopes are involved, is most quantitative under steady state conditions. Therefore, clamps should be of a duration (~2 h) that is sufficient for the actions of insulin to obtain steady state.  The section that follows describes, a) the technical procedures used on the day of a clamp experiment; b) preparation of insulin and tracer infusates; and c) analyses of plasma and tissue radioactivity.  Familiarity with these procedures combined with laboratory demonstration provides the investigator with the necessary background to clamp the conscious mouse. </div></div>

[STEPS]
?. Surgical catheterization of the carotid artery and jugular vein in mice at least 5 days prior to the day of the study (refer to protocol for Surgical Catheterization of the Carotid Artery and Jugular Vein).
?. Weigh mouse and start fast (suggested starting time between 7:00 and 8:00 AM) by placing mouse in a plastic container with fresh bedding.
?. Set up microrenathane tubing to swivel and connectors as needed (see Figure 1).
?. Collect donor blood (Reagent 1).
?. Prepare tracers, if needed (Reagent 3, Reagent 4).
?. If using [3-³H]glucose, draw tracer infusate into a 1 ml syringe and place it securely into the pump slot.
?. Fill the arterial line with heparinized saline.  Leave the sampling syringe connected to the top of the swivel.  Fill the venous line with non-heparinized saline (or tracer, if using [3-³H]glucose).  Plug the free end of the 3 cm microrenathane tubing going into the venous port of the swivel (A in Figure 1) or the free end of the 3 cm tubing going into the 3-way connector if using [3-³H]glucose (B in Figure 1) with a stainless steel plug.  Filling these lines prevents infusion of air into the mouse.
?. If using [3-³H]glucose, weigh the mouse and hook it up to the microrenathane tubing ~100 minutes prior to clamp start as shown in Figure 1.
?. Begin baseline primed-continuous tracer infusion at least 90 minutes prior to clamp start.  We typically use a 1.2 μCi prime followed by a 0.04 μCi/min continuous infusion.
?. Prepare Protocol sheets using mouse weight, insulin dose, sample times and volumes.
?. Prepare insulin (Reagent 2).
?. Once donor blood and insulin are prepared, draw glucose infusate (50% dextrose mixed with or without [3-³H] glucose), insulin infusate, and donor blood into separate 1 ml syringes and place them securely into the pump slots.
?. Connect 10 cm microrenathane tubing to each infusate syringe, and connect tubing to a 4-way connector (see Figure 1).  Fill each line with glucose, insulin, and donor blood infusates up to the 4-way connector.
?. Once the last basal sample is taken, stop the baseline tracer infusion pump if using [3-³H]glucose and connect the 4-way connector to the 3 cm microrenathane tubing connected to the infusion port of the swivel, and begin infusion of donor blood.
?. Once donor blood reaches the mouse, start study by beginning insulin and glucose infusion.
?. To take blood samples for analysis of plasma metabolites: ♦  Insert a blunt syringe into the free end of the arterial line prior to releasing the clamp.   ♦  Release the clamp and draw the desired volume of blood into the syringe. ♦  Clamp the arterial line and remove the syringe.  ♦  Dispense the blood from the syringe into a 0.5 ml EDTA tube and mix by “flicking” the tube. ♦  Centrifuge for 1 min at 16,000 g.  Transfer the plasma to an appropriately labeled 1.5 ml tube.  Plasma can be temporarily stored on ice but should be permanently stored at –20ºC. ♦  While the blood is being centrifuged, insert the sampling syringe back into the arterial line.  Pull back slightly on the plunger to draw any air bubbles into the sampling syringe.  Very slowly, give back the ~ 20 – 40 μl of blood originally taken such that the arterial line becomes lightly red to clear.
?. Analysis of radioactivity (when applicable) Plasma Glucose and 2-Deoxyglucose Radioactivities Reagents and supplies: Saline Saturated benzoic acid (3.4 g/l) 0.3 N Ba(OH)² diluted 1:3 with ddH²O. Prepared fresh.  0.3 N ZnSO4 diluted 1:3 ml with ddH²O. Prepared fresh. 1.5 ml tubes 20 ml borosilicate scintillation vialsProcessing 14C- or 3H-2-deoxyglucose standards: 1.    Add 196 μl ddH²O to 4 μl of standard (saved from bolus of 2-deoxyglucose) and vortex. 2.    Pipette 50 μl diluted standard into a 1.5 ml tube and add 950 μl ddH²O. 3.    Pipette 100 μl into scintillation vial. 4.    Add 900 μl ddH²O and 10 ml scintillation fluid.  5.    Measure radioactivity in a liquid scintillation counter. Processing [3-3H]glucose standards:1.    Dilute tracer standards (both for baseline and the clamp period) 1:200 in saturated benzoic acid (5  μl in 995 μl). 2.    Add 10 μl of this diluted standard directly to each of three scintillation vials labeled CS.  Add 990 μl ddH²O and cap. 3.    Add 10 μl of the diluted standard directly to each of three separate scintillation vials labeled CSE and set aside for drying later (see Plasma processing below). 4.    Add 10 μl of the diluted standard to each of three 1.5 ml tubes. To each tube add 10 μl saline + 100 μl diluted Ba(OH)2 (prior to ZnSO4) + 100 μl diluted ZnSO4. 5.    Vortex and spin down for 5 minutes at 16,000 g. 6.    Transfer 100 μl of the supernatant to each of three scintillation vials labeled CRS and set aside to dry later (see Plasma processing below).Plasma processing: 1.    Add 10 μl of plasma sample and 10 μl saline to a 1.5 ml tube. 2.    Add 100 μl diluted Ba(OH)²solution and 100 μl diluted ZnSO4 (add Ba(OH)² prior to ZnSO4). 3.    Vortex and centrifuge (5 min at 16,000 g). 4.    Pipette 100 μl of supernatant into scintillation vial. 5.    Dry in oven with a trap for ³H²O.  Include standards (CSE and CRS) for drying at this time.a,b6.    Add 1 ml ddH2O and 10 ml scintillation fluid. 7.    Measure radioactivity in a liquid scintillation counter.         a.   To determine ³H²O accumulation, a measure of glycolytic rate, pipette 50 µl of the supernatant into a second set of vials and do not dry. ³H²O will be the difference in radioactivity between these non-dried samples and the dried samples (correcting for the difference in the volume of supernatant used).          b.    Analysis of samples containing only radioactive 2-deoxyglucose does not require that they be dried.  Simply add 900 ml ddH²O and 10 ml scintillation fluid to the 100 μl of supernatant in the scintillation vial.  Tissue 2-Deoxyglucose Phosphate RadioactivityReagents and supplies: 0.3N  Ba(OH)2       0.3N  ZnSO4 0.5% perchloric acid        Liquid  nitrogen              Ice       20 ml borosilicate scintillation vials 5 ml and 13 ml large polypropylene tubes w/ caps 1.5 ml tubes 5N KOH and 10 N HCl        Forceps              Homogenizer              pH  met1.    Keep tissues and 5 ml tubes in liquid nitrogen and make sure that the end of the forceps that will come in contact with tissue is cooled in liquid nitrogen. 2.    Weigh each tissue sample (~60 mg), transfer to 5 ml tube in liquid nitrogen and record weight. Note: you will not obtain 60 mg in all tissues (e.g. soleus muscle). In those cases, simply weigh out the entire amount of tissue obtained.  3.    For each sample, place on ice the 5 ml tube containing the tissue and homogenize in 1.5 ml 0.5% perchloric acid.  Keep the homogenized sample on ice while the remaining tissues are homogenized. 4.    Centrifuge tubes for 20 min at 2000 g at 4°C. 5.    Place tubes on ice and transfer 1.25 ml of the supernatant to 13 ml tubes.  If you are unable to extract 1.25 ml of supernatant, note the volume of supernatant you are able to extract. 6.    Neutralize each sample (pH ~7.5) using KOH and HCl. Record the total volume of KOH and HCl used to neutralize each sample. 7.    Centrifuge tubes for 10 min at 2000 g at 4°C.8.    Label two scintillation vials (A and B) for each tissue, and number consecutively         For the A vials (measurement of 2-deoxyglucose phosphate and 2-deoxyglucose)                Add 125 µl of neutralized supernatant and 875 µl ddH²O          For the B vials (measurement of 2-deoxyglucose only)                 Pipette 250 µl of neutralized supernatant into 1.5 ml tubes                 Add 125 µl of stock 0.3N BaOH                 Add 125 µl of stock 0.3N ZnSO4                Centrifuge at 16,000 g for 5 minutes                 Add 250 µl of supernatant and 750 µl ddH²O to vials9.     Add 10 ml of scintillation fluid to each vial, shake and measure radioactivity in liquid scintillation counter. 10.   Deoxyglucose phosphate is the difference between radioactivity in A and B.