Leading researchers and clinical investigators describe their best cutting-edge techniques for studying, at both the molecular and biochemical levels, the defects in insulin production and action associated with diabetes. Written in step-by-step detail to ensure ready reproducibility and robust results, these techniques allow investigators, both novices and those already active in the field, to study every major facet of insulin production and action. Each protocol includes an introduction to the technique, an explanation of its application, and a list of materials. Practical notes discuss how to avoid pitfalls, as well as how to adapt the methods to your own research.

Part I. Insulin Production

Isolation of Islets of Langerhans from Rodent Pancreas
Colleen B. Kelly, Libby A. Blair, John A. Corbett, and Anna L. Scarim

Purification of Rat Pancreatic b-Cells by Fluorescence-Activated Cell Sorting
Geert Stangé, Mark Van De Casteele, and Harry Heimberg

Assessment of Insulin Secretion in the Mouse
Marcela Brissova, Wendell E. Nicholson, Masakazu Shiota, and Alvin C. Powers

Detection of Insulin Production by Immunohistochemistry
Susan C. Campbell and Wendy M. Macfarlane

Quantification of the Level of Insulin Gene Expression
Susan C. Campbell and Wendy M. Macfarlane

Chromatin Immunoprecipitation Using Isolated Islets of Langerhans
Marcelina Párrizas, Sylvia F. Boj, Reini F. Luco, Miguel Angel Maestro, and Jorge Ferrer

Adenoviral Gene Transfer into b-Cell Lines
Amber L. Mosley and Sabire Özcan

Utilization of NOD Mice in the Study of Type 1 Diabetes
Dennis G. Karounos and Susan E. Goes

Part II. Insulin Action

Introduction of DNA into 3T3-L1 Adipocytes by Electroporation
Shuichi Okada, Masatomo Mori, and Jeffrey E. Pessin

Analysis of Insulin-Stimulated Glucose Uptake in Differentiated 3T3-L1 Adipocytes
Jaganathan Lakshmanan, Jeffrey S. Elmendorf, and Sabire Özcan

Fractionation Analysis of the Subcellular Distribution of GLUT-4 in 3T3-L1 Adipocytes
Jeffrey S. Elmendorf

Visualization and Quantitation of Integral Membrane Proteins Using a Plasma Membrane Sheet Assay
John B. Knight and Ann Louise Olson

Assaying Tyrosine Phosphorylation of Insulin Receptor and Insulin Receptor Substrates
Eileen L. Whiteman and Morris J. Birnbaum

Measuring Insulin-Stimulated Phosphatidyl-Inositol 3-Kinase Activity
Li-Ping Wang and Scott A. Summers

Assaying AKT/ Protein Kinase B Activity
Kyle L. Hoehn and Scott A.Summers

Measurements of Cellular Phosphoinositide Levels in 3T3-L1 Adipocytes
Daryll B. DeWald

Measurement of Glycogen Synthesis and Glycogen Synthase Activity in 3T3-L1 Adipocytes
Matthew J. Brady

Measurement of Contraction-Stimulated GLUT-4 Translocation in Isolated Skeletal Muscle
Joseph T. Brozinick, Jr.

Single Embryo Measurement of Basal- and Insulin-Stimulated Glucose Uptake
Maggie M-Y. Chi and Kelle H. Moley

Immunohistologic Staining of Muscle and Embryos to Detect Insulin-Stimulated Translocation of Glucose Transporters
Mary O. Carayannopoulos and Kelle H. Moley

Index