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Test Name

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Industry Price

Y4001

Hyperinsulinemic-euglycemic clamp experiments

$100

$300

The surgery is performed at 4-5 days prior to the hyperinsulinemic-euglycemic clamp to establish a chronic catheter for intravenous infusion of substances (e.g., glucose, insulin) during the clamp. For this, a mouse is anesthetized with an intraperitoneal injection of ketamine and xylazine, and a catheter is inserted in the right jugular vein. On the day of clamp experiment, an overnight-fasted mouse is placed in an over-sized restrainer (i.e., rat-sized) for the experiment to be conducted in awake and minimally-stressed state. The tail is tethered using a tape for 2 hours prior to the start of experiment for acclimatization. A 3-way connector is attached to the jugular vein catheter for intravenous infusion, and the blood samples are obtained from the tail vessels requiring a small tail cut. A 2-hour hyperinsulinemic-euglycemic clamp is conducted with a primed-continuous infusion of human insulin at a rate of 15 pmol/kg/min to raise plasma insulin within a physiological range (~300 pM). Blood samples (20 ml) are collected at 10-20 min intervals for the immediate measurement of plasma glucose concentration, and 20% glucose is infused at variable rates to maintain glucose at basal concentrations (~6 mM). Insulin-stimulated whole body glucose metabolism is assessed with a continuous infusion of [3-³H]glucose (0.1 mCi/min) throughout the clamps. Basal rates of whole body glucose turnover are assessed using a primed-continuous infusion of [3-³H]glucose for 2 hours prior to the start of clamp. All infusions are performed using the microdialysis pumps, and all procedures are approved by Yale University Animal Care and Use Committee. To estimate insulin-stimulated glucose uptake in individual tissues, 2-deoxy-D-[1-¹⁴C]glucose (2-[¹⁴C]DG) is administered as a bolus (10 mCi) at 75 min after the start of clamp. Blood samples (20 ml) are taken at -5, 80, 85, 90, 100, 110, and 120 min of clamp for the measurement of plasma [³H]glucose, ³H₂O, and/or 2-[¹⁴C]DG concentrations. Additional blood samples (20 ml) are collected before and at the end of clamp for the measurement of plasma insulin concentrations. At the end of clamp, mouse is anesthetized with sodium pentobarbital injection, and tissues are taken and stored for biochemical/molecular analysis. The clamp experiment measures tissue-specific insulin action and glucose metabolism and includes the following measurements: 1) basal and insulin-stimulated hepatic glucose production, 2) insulin-stimulated whole body glucose uptake, glycolysis, and glycogen plus lipid synthesis, and 3)insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis in individual tissues (e.g., skeletal muscle, adipose tissue, heart). Additionally, biochemical/molecular assays may be performed to assess tissue-specific insulin signaling activities (e.g., insulin-stimulated tyrosine phosphorylation of IRS, PI 3-kinase activity) and tissue-specific triglyceride contents. Further details of the clamp experiment can be found in the following references: Diabetes 53:1060 (2004), J. Clin. Invest. 114:823 (2004).

Y4002

Hyperglycemic clamp experiments

$80

$240

The procedure involves chronic cannulation of the jugular vein which is necessary for The surgery is performed at 4-5 days prior to the hyperglycemic clamp to establish a chronic catheter for intravenous infusion of substances (e.g., glucose, insulin) during the clamp. For this, a mouse is anesthetized with an intraperitoneal injection of ketamine and xylazine, and a catheter is inserted in the right jugular vein. On the day of clamp experiment, an overnight-fasted mouse is placed in an over-sized restrainer (i.e., rat-sized) for the experiment to be conducted in awake and minimally-stressed state. The tail is tethered using a tape for 2 hours prior to the start of experiment for acclimatization. A 3-way connector is attached to the jugular vein catheter for intravenous infusion, and the blood samples are obtained from the tail vessels requiring a small tail cut. A 2-hour hyperglycemic clamp is conducted with a variable infusion of 20% glucose to raise and maintain plasma glucose concentrations at ~16 mM. Blood samples (20 ml) are collected at 10-20 min intervals for the immediate measurement of plasma glucose concentrations using Beckman Glucose Analyzer. The area under curve of plasma glucose and insulin profiles is assessed to determine glucose-induced insulin secretion in vivo (i.e., pancreatic ß-cell function).