References

Lomedico, P., Rosenthal, N., Efstratidadis, A., Gilbert, W., Kolodner, R., and Tizard, R. (1979) The structure and evolution of the two nonallelic rat preproinsulin genes. Cell 18, 545-558. 2. Bell, G. I., Swain, W. F., Pictet, R., Cordell, B., Goodman, H. M., and Rutter, W. J. (1979) Nucleotide sequence of a cDNA clone encoding human preproinsulin. 3. Clark, P. M. (1999) Assays for insulin, proinsulin(s) and C-peptide. Ann. Clin. Biochem. 36, 541-564. 4. Gray, I. P., Siddle, K., Frank, B. H.,...

Assay of Glycogen Synthesis Rate

Insulin-stimulated glycogen synthesis in 3T3-L1 adipocytes is dependent on increased glucose uptake and glycogen synthase activity. The cells are pre-treated with insulin to activate both processes before the addition of 14C-labeled glucose to the extracellular media. The cells are washed and lysed, and cellular glycogen is precipitated with ethanol. Glycogen pellets are washed and dried overnight, and 14C-glucose incorporation into glycogen is measured by liquid scintillation counting. 1. Use...

Geert Stange Mark Van De Casteele and Harry Heimberg Introduction

The b-cell is receptive to intricate hormonal, neuronal and nutrient signaling which is key for normal physiology but complicates the study of specific effects of individual factors on b-cell function. To preserve the microenvironment of the b-cell, most studies of b-cell physiology have been performed on in vitro cultured islets of Langerhans. However, whereas islets in the pancreas are highly vascularized and oxygenated, ischemic conditions cannot be avoided in the center of cultured isolated...

Alex Kolesar Micrograph

Preparation of plasma membrane PM sheets from intact cells is a simple method for obtaining an immobilized cytoplasmic PM surface that can be probed with antibodies or chemical probes. This method was first reported by Robinson et al. 1 who used it to detect the presence of the GLUT-4 glucose transporter at the PM as a result of insulin stimulation. We have extended this technique in order to use it as a means to quickly and efficiently obtain a highly purified plasma membrane fraction 2 . We...

Deoxyglucose Uptake Assay

Blastocysts are moved by mouth pipet to HTF media supplemented with D-glucose to give a final concentration of 5.6 mM and containing 500 nM insulin for 30 min. Following this preincubation, they are moved in groups of 10-20 into Ringers wash and then into media containing 200 iM 2-DG for 15 min. A basal group is placed in HTF with 5.6 mM D-glucose with no insulin added. 2. The embryos are then removed, washed in DG-free, BSA-free Ringers solution for 1 min, and then transferred with 0.5-1 L...

Introduction

Insulin gene expression levels can be detected and quantified using a broad range of molecular biology techniques. Traditionally, detection of insulin mRNA levels in both b-cell lines and purified islets has been performed by Northern blot analysis, using the full-length insulin cDNA as a labeled probe 1 . This technique is described here, and is still the most useful method for detection of endogenous insulin mRNA levels densitometry being applied to allow quantification of band intensities ....

Jeffrey S Elmendorf Introduction

In 1980, two groups simultaneously provided evidence of the existence of an intracellular pool of glucose transporters in rat adipocytes 1,2 . We now know that facilitative glucose uptake occurs through a family of highly related integral membrane proteins that share significant sequence similarity. Of the established glucose transporter isoforms, GLUT-4 is highly expressed in adipose tissue and striated muscle 3 . In the basal state, GLUT-4 cycles slowly between the plasma membrane and one or...

Assaying Insulin Levels Utilizing an Enzyme Linked Immunoassay

Because the rodent insulin sequence is identical for rats and mice, the rat assay kits are used for both insulin and C-peptide measurements see Note 7 1. Collect blood by venopuncture and allow to clot. 2. Centrifuge for separation of sera see Note 8 . 3. Prepare 96-well plate for standards, controls, and samples to be run in duplicate. Pipet 25 iL each of standards, controls, and samples into designated wells see Note 9 . 4. Add 50 iL peroxidase-conjugated mouse monoclonal anti-insulin working...

Materials Reagents

Perfusion of the Mouse Pancreas In Situ 1. Krebs-Ringer bicarbonate buffer KRB 4.4 mM KCl, 2.1 mM CaCl2, 1.5 mM KH2PO4, 1.2 mM MgSO4, 29 mM NaHCO3, and 116 mM NaCl prepared on the day prior to perfusion. 2. Dextran-70 cat. no. 17-0280-02, Amersham . Prepare a 3 w v solution in KRB to form KRB-dextran on the day prior to perfusion. 3. Nembutal sodium solution 50 mg mL cat. no. NDC-0074-3778-04, Abbott diluted 1 10 in 0.9 NaCl. Store up to 1 mo at 48C. 4. Dextrose cat. no. BP350-1000,...

Islet Purification from Rodent Pancreas

Ficoll Gradient Islet

The same general protocol is used for the purification of islets from mouse and rat pancreata. All media used for islet isolation should be equilibrated to room temperature except the HBSS. Note that this entire procedure, excluding centrifugation and digestion, should be performed using sterile technique. 1. A 5-rat or 25-mouse preparation will require a minimum of 500 mL HBSS, a maximum of 500 mL cCMRL-1066, Ficoll dilutions 20 mL of 25 dilution and 10 mL of 23 , 20.5 , and 11 dilutions , and...

Adenoviral Gene Transfer into Insulinoma Cell Lines

Insulinoma Cell Line

In order to optimize the infection of insulinoma cell lines, it is important to test the efficiency of each constructed recombinant adenovirus individually. Efficiency of gene transfer can vary based on the stability and toxicity of the expressed gene product itself in insulinoma cells. The adenoviral gene transfer Fig 1. Analysis of the infection efficiency using different titers of the GFP adenovirus. A MIN6 cells infected with various titers of the GFP adenovirus. The adenoviral titer is...

Mouse Insulin Immunoassay of Samples Acquired During Perfusion of the Mouse Pancreas In Situ

Insulin Tubes

An example of insulin secretion data from the in siiw-perfused mouse pancreas is shown in Fig. 4. 1. Label duplicate plain tubes in the sequence T total counts and N nonspecific binding . 2. Label duplicate antibody-coated tubes in the sequence R reference or maximum binding , 4-10 reserved for standard insulin , and 11-n n-number of samples . Put tubes in wire racks. 3. Pipet 0.1 mL RIA buffer into tubes T-R . 4. Pipet 0.1 mL each insulin standard solution into tubes 4-10 beginning with 0.25...

Mouse Insulin Immunoassay of Plasma Acquired During Glucose Tolerance Testing in Wild Type or Transgenic Mice

An example of insulin secretion during glucose tolerance testing in mice is 1. Label duplicate plain 12 X 75-mm tubes in the sequence T , N , R , and 4-n . 2. Pipet 0.1 mL RIA buffer into tubes N and R . Pipet 0.1 mL of each standard solution into tubes 4-9 , beginning with 5 pg mL into tube 4 . 3. Pipet 0.1 mL each diluted plasma sample into the remaining tubes, beginning with the first sample into tube 10 . 4. Add 0.1 mL diluted primary antibody repeating pipet to all tubes except T and N ....

Mini Protean Ii

It should be noted that muscles can also be stimulated in situ via the innervating nerve or by acute exercise and then removed and placed in incubation for measurement of GLUT-4 translocation. In the first case, the muscle is stimulated by 200-ms trains of stimuli at a frequency of 70 Hz, with each impulse in a train being 0.1 ms. The trains are delivered one per second at 10-15 V for 2 X 10 min with a 1-min rest in between. For acute exercise, rats can run on a treadmill at 20 m min for 1 h...

Perfusion of the Mouse Pancreas In Situ

Perfusion Mouse Diagram

Diagrams of the perfusion chamber and mouse surgery are shown in Fig. 2 1. Attach a three-way stopcock to the O2 CO2 flow regulator with a 5-cm section of 4.8-mm-I.D. tubing. Connect three T-shaped connectors end-to-end with 5-cm sections of 4.8-mm-I.D. tubing. Connect one L-shaped connector to one free end of the T's. Connect approx 2 m of 4.8-mm-I.D. tubing to the remaining free end of the T's for later attachment to stopcock-flow regulator assembly. Fig. 3. Perfusion of mouse pancreas in...

Insulitis Grading

One of the hallmarks of type 1 diabetes is the development of insulitis lymphocytic infiltration of the islets. Grading the severity of the insulitis is a useful immunological parameter to monitor when evaluating immunological therapies or genetic influences. 1. Mice are killed by CO2 inhalation. All four legs are pinned down on a Styrofoam board and the abdomen is cleaned with 70 ethanol. 2. Using forceps, an incision through the fur but not into the peritoneal cavity is made just below the...

Rat Pancreas Isolation

Bile Duct Cannulation Rat

The following procedure is used for isolating pancreata from rats weighing 150-300 g. For optimal success, the procedure should be performed as quickly as possible to avoid tissue degradation. This protocol can be used to isolate pan-creata from one to five rats during a single isolation. It is not recommended to use more than five rats per isolation because of the extended time for pancreas removal. The protocol anticipates that the total surgery time will be no longer than 30 min for a...

Eileen L Whiteman and Morris J Birnbaum Introduction

This chapter describes techniques to successfully detect tyrosine phosphorylation of the insulin receptor and insulin receptor substrate proteins. These assays demonstrate whether the insulin signaling pathway is activated at its earliest stages. The insulin receptor IR is a transmembrane glycoprotein whose subunits are joined by disulfide bonds to create an a2b2 heterotetramer. Its extracellular a-sub-units constitute the binding domain for insulin. The transmembrane and intracellular...

Mary O Carayannopoulos and Kelle H Moley Introduction

The facilitative glucose transporters constitute a family of integral membrane proteins, each with different substrate specificities, tissue distribution, and transport kinetics. It is possible to localize and track movement of facilitative glucose transporters within a cell using several different techniques subcellular fractionation 1,2 , surface labeling with the bis-mannose photolabel or Holman's reagent 3-5 , immunoelectron microscopy 6,7 , immunohistological techniques using confocal...