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Hyperinsulinemic-euglycemic clamp experiments

Common Name(s): Hyperinsulinemic-euglycemic clamp

Center	Yale University School of Medicine
Center Core	Metabolic Phenotyping Core
Test No.	Y4001
Test Price	$600.00
Description	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).
Data	No data will be collected for this catalog item
Keywords	clamp, diabetes, glucose, insulin, insulin action, insulin resistance, insulin secretion
Tissues	pancreas

Name	Options
Carbohydrate Metabolism
Diabetes
Insulin and Insulin Function
Metabolism and Metabolomics

Name	Abbreviation	Options
glucose uptake	None
average clamp glucose	None
fat body mass	None
lean body mass	None
total body weight	None
glucose, basal	None
hepatic glucose production, basal	None
glucose synthesis	None
glucose turnover, whole body	None
glycogen/lipid synthesis, whole body	None
glycolysis, whole body	None
glycogen synthesis	None
glycolysis	None

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