Evaluation of Glucose Homeostasis

Sami Heikkinen1, Carmen A. Argmann2, Marie‐France Champy3, Johan Auwerx4

1 A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, 2 Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, 3 Institut Clinique de la Souris, Illkirch, 4 Institut de Genetique et de Biologie Moleculaire et Cellulaire and Institut Clinique de la Souris, Illkirch
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 29B.3
DOI:  10.1002/0471142727.mb29b03s77
Online Posting Date:  January, 2007
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Abstract

Obesity and dyslipidemia are often found in association with insulin resistance (IR). These components combined with hypertension characterize the most common endocrine disorder in humans, the metabolic syndrome. Thus, in addition to profiling body weight evolution and lipid metabolites, glucose tolerance (a reflection of IR) and insulin sensitivity should also be considered as part of any metabolic phenotyping protocol. The ability to measure IR and glucose tolerance is important not only in the quest to fully understand the pathogenesis of the metabolic syndrome in the mouse, but also to test the effects of potential interventions. This unit presents a variety of tests used for this purpose, including direct blood glucose measurements, insulin measurement by ELISA, the homeostatic model assessment, glucose tolerance and insulin sensitivity tests, and the euglycemic clamp.

Keywords: metabolic phenotyping; insulin resistance; glucose tolerance; hyperinsulinemic‐euglycemic clamp; mice

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Glucose Measurement by Blood Glucose Monitor
  • Basic Protocol 2: Insulin Measurement by ELISA
  • Basic Protocol 3: Homeostatic Model Assessment
  • Basic Protocol 4: Meal Tolerance Test
  • Basic Protocol 5: Oral Glucose Tolerance Test
  • Basic Protocol 6: Intraperitoneal Insulin Sensitivity Test
  • Basic Protocol 7: Intraperitoneal Glucose Tolerance Test
  • Basic Protocol 8: Hyperinsulinemic‐Euglycemic Clamp
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Glucose Measurement by Blood Glucose Monitor

  Materials
  • Mice
  • Low‐ and high‐level glucose control solutions (e.g., Accu‐Chek active glucose control solutions, Roche Diagnostics; low concentration: 50 mg/dl, reference range 42 to 72 mg/dl, and high concentration: 300 mg/dl, reference range 290 to 328 mg/dl)
  • Blood glucose monitor and associated glucose test strips for glucose measurement (e.g., Accu‐Chek Active, Roche Diagnostics)
  • Scalpel blade

Basic Protocol 2: Insulin Measurement by ELISA

  Materials
  • Ultrasensitive Mouse Insulin ELISA kit (e.g., Mercodia) containing:
    • 96‐well microtiter plates coated with mouse monoclonal anti‐insulin antibody
    • Calibrators (i.e., standards)
    • Antibody‐enzyme conjugate (concentrate) and buffer
    • Wash buffer (concentrate)
    • TMB substrate reagent (3′,3′,5′,5′‐tetramethylbenzidine; light sensitive)
    • Stop solution: 0.5 M H 2SO 4
  • Mouse serum or plasma
  • 0.9% (w/v) NaCl
  • Microtiter plate shaker
  • Microtiter plate washer (optional)
  • Plate reader capable of measuring at 450 nm

Basic Protocol 3: Homeostatic Model Assessment

  Materials
  • Mice
  • Heparinized tubes for blood sampling (e.g., Microvette, Sarstedt)
  • Mouse cages
  • Scalpel blade
  • Refrigerated microcentrifuge
  • Additional reagents and equipment for measuring blood glucose (see protocol 1) and insulin (see protocol 2)

Basic Protocol 4: Meal Tolerance Test

  Materials
  • Mice
  • Isoflurane inhalation anesthesia unit (composed of a mixing and flow‐control system for O 2/isoflurane anesthesia; e.g., TEM, http://www.TEM.fr)
  • Non‐heparinized glass capillaries, 50‐ to 100‐µl (e.g., Vitrex by Modulohm)
  • Mouse chow
  • Mouse cages
  • Refrigerated microcentrifuge
  • Additional reagents and equipment for measuring blood glucose (see protocol 1) and insulin (see protocol 2) levels

Basic Protocol 5: Oral Glucose Tolerance Test

  Materials
  • Mice
  • 20% (w/v) aqueous glucose solution (see recipe), sterile
  • Mouse cages
  • Animal scale
  • Blood glucose monitor and associated glucose test strips for glucose measurement (e.g., Accu‐Chek Active, Roche Diagnostics)
  • 1‐ml syringes (e.g., Terumo) and 22‐G ball‐tip needle (e.g., Popper and Sons)
  • Scalpel blade
  • Timer

Basic Protocol 6: Intraperitoneal Insulin Sensitivity Test

  Materials
  • Mice
  • Insulin solution for IPIST (see recipe)
  • Mouse cages
  • Animal scale
  • 1‐ml syringes (e.g., Terumo) and 25‐G × 5/8‐in. needles (e.g., Terumo)
  • Blood glucose monitor and associated glucose test strips for glucose measurement (e.g., Accu‐Chek Active, Roche Diagnostics)
  • Scalpel blade
  • Timer

Basic Protocol 7: Intraperitoneal Glucose Tolerance Test

  Materials
  • Mice
  • Anesthesia: ketamine and xylazine
  • Insulin solution for euglycemic clamp (see recipe)
  • 20% (w/v) glucose in saline solution (see recipe), sterile
  • Saline: 0.9% (w/v) NaCl
  • 70% (v/v) ethanol
  • Mouse femoral catheter (e.g., M‐FAC, Braintree Scientific)
  • Mouse cages with flat‐grated lid
  • Animal scale
  • 1‐ and 2‐ml syringes
  • Single syringe infusion pumps capable of holding a 2‐ml syringe with a range of infusion rates of 5 to 20 µl/min (e.g., Harvard Apparatus or TSE Systems)
  • Multi‐syringe infusion pump capable of holding a minimum of six individual 1‐ml syringes with a range of infusion rates of 3 to 10 µl/min (e.g., Harvard Apparatus or TSE Systems)
  • Tubing (e.g., Tygon tubing, Ø 0.51 mm, Biorad)
  • Tubing connectors (e.g., 23‐G needles, Terumo)
  • Y‐shaped canula connector (e.g., C312ICP/O, Protech International)
  • Retort stand with a movable arm to hold the Y‐shaped canula connector and tubing in place
  • Timer
  • Blood glucose monitors and associated glucose test strips for glucose measurement (e.g., Accu‐Chek Active, Roche Diagnostics)
  • Scalpel blade
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Figures

Videos

Literature Cited

Literature Cited
   Ayala, J.E., Bracy, D.P., McGuinness, O.P., and Wasserman, D.H. 2006. Considerations in the design of hyperinsulinemic‐euglycemic clamps in the conscious mouse. Diabetes 55:390‐397.
   Champy, M.F., Selloum, M., Piard, L., Zeitler, V., Caradec, C., Chambon, P., and Auwerx, J. 2004. Mouse functional genomics requires standardization of mouse handling and housing conditions. Mamm. Genome 15:768‐783.
   Hedrich, H. (ed.) 2004. The Laboratory Mouse. Elsevier Academic Press, London.
   Levy, J.C., Matthews, D.R., and Hermans, M.P. 1998. Correct homeostasis model assessment (homa) evaluation uses the computer program. Diabetes Care 21:2191‐2192.
   Matthews, D.R., Hosker, J.P., Rudenski, A.S., Naylor, B.A., Treacher, D.F., and Turner, R.C. 1985. Homeostasis model assessment: Insulin resistance and beta‐cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 28:412‐419.
   Michael, M.D., Kulkarni, R.N., Postic, C., Previs, S.F., Shulman, G.I., Magnuson, M.A., and Kahn, C.R. 2000. Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction. Mol. Cell 6:87‐97.
   Wallace, T.M., Levy, J.C., and Matthews, D.R. 2004. Use and abuse of homa modeling. Diabetes Care 27:1487‐1495.
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