References

D. (1993). The glucose transporter family structure, function and tissue-specific expression. Biochem. J. 295, 329-341. 2. Cushman, S. W. and Wardzala, L. J. (1980). Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell apparent translocation of intracellular transport systems to the plasma membrane. J. Biol. Chem. 255(10), 4758-4762. 3. Goodyear, L. J., King, P. A., Hirshman, M. F., Thompson, C. M., Horton, E. D., and Horton, E....

Notes

It is important to proceed as soon as possible with the digestion therefore, the dissection should be done fast. On average, one person should be able to process five rats within 30 min. 2. It is of crucial importance to test different batches of collagenase for their yield and toxicity. Therefore, the number of b-cells that survive the isolation and can be kept in culture without losing their functional responsiveness is determined by the quality of the collagenase. The concentration of the...

Insulin Therapy

For short-term studies, insulin therapy is usually not necessary and diabetic mice can live for 4 wk after diagnosis without insulin treatment. For long-term studies, mice can be treated with long-acting insulins such as PZI Beef and Pork Insulin. 1. Treat NOD mice with PZI Beef and Pork Insulin 0.5-1 U d or NPH. For NPH insulin, we use human recombinant insulin diluted in insulin diluent 1 10 administered at a dose of 0.125-1.00 U d (0.1 mL) using a 27-gauge needle in a 0.5-mL insulin syringe...

Accelerating the onset of Diabetes

NOD mice develop spontaneous diabetes between 20 and 50 wk of age. Investigators have found that administration of 1-2 doses of cyclophosphamide can dramatically accelerate the onset of diabetes so that the peak onset of diabetes is 30-40 d rather than 30-40 wk (4) (see Note 3). 1. Cyclophosphamide is administered as a single ip injection, 100-300 mg kg body weight, to mice older than 7 wk of age (4). 2. Mice are then monitored daily for glycosuria. Once glycosuria is detected, the diagnosis of...

Adoptive Transfer

The adoptive transfer technique is not only an accelerated model of spontaneous diabetes but it also allows the investigator to evaluate the cellular and genetic components involved in the immunopathogenesis of diabetes. As an accelerated model of diabetes, recipient mice develop diabetes 4-12 wk after transfer of donor cells compared to 20-40 wk of age in the spontaneous disease models (7-9). The critical factors are to maintain a genetically susceptible host and to give adequate numbers of...

Matthew J Brady Introduction

Insulin stimulates the storage of glucose as glycogen in muscle and adipose tissue through the coordinate increase in glucose uptake and modulation of glycogen metabolizing enzymes (1). Insulin binds to its receptor in peripheral tissues and initiates several signaling cascades to increase glucose uptake via translocation of the glucose tranporter-4 (GLUT-4) containing vesicles to the plasma membrane (see Fig. 1). Glucose enters the cells and is phosphorylated by hexokinases to form...

Muscle Preparation

Male B6 X SJL F1 mice are allowed free access to food and water and kept on a 12 12-h light-dark cycle. The mice are then given an intraperitoneal injection of glucose (2 g kg of body weight) and either insulin (6 U) or saline. 2. Over the next 30 min, the mice are anesthetized with pentobarbital (5 mg 100 g of body weight), and a butterfly needle connected to iv tubing is introduced in the dorsal tail vein of the animal. At exactly 30 min after the insulin or saline injection, 5...

Animal Care

When handling the NOD mice, use the following procedures 1. Wear laboratory coat, protective sleeves, gloves, and face mask. 2. All work is performed in a HEPA-filtered laminar-flow workbench. The surface is precleaned with sterilant (Spor Klenz, 1 ). 3. For the mice to maintain a high incidence of spontaneous diabetes, they should be housed in a murine-specific pathogen-free animal facility. The cleaner the facility, the higher the incidence of spontaneous diabetes. 4. NOD mice are housed in...

Deoxyglucose Assay Using Enzymatic Cycling in Oil Wells

The basic enzymatic cycling reaction involves three steps in order to measure deoxyglucose. In the first step, a 0.1- to 0.2- L aliquot is removed from the neutralized acid extraction and used in the specific reaction sequence, ending in reduction of a pyridine nucleotide. This reaction sequence is found in Fig. 1 and described in detail here. The second step is the enzymatic cycling or amplification step. NADPH is alternatively oxidized and reduced as seen in Fig. 2. In each oxidation...

Equipment

Microisolater cages (Lab Products, Inc., 742 Sussex Ave., Seaford, DE). 2. Glucose meter (Bayer Elite XL http www.glucometerstore.com and http www. 3. Microhematocrit capillary tube. 5. Scalpel with fine 11 blade. 6. Insulin syringe with 27-gauge needle. 7. Heparinized capillary tube for plebotomy. 8. HEPA-filtered laminar-flow workbench.

Fractionation

Disposable cell scraper (Fisher Scientific, Hanover Park, IL, cat. no. 08-773-2). 2. 10 mL BD Brand syringe (Fisher Scientific, cat. no. 14-823-2A). 3. Needles, BD Brand, 22G1 (Fisher Scientific, cat. no. 14-826B). 4. HEPES-EDTA-sucrose (HES) buffer 20 mM HEPES, 1 mM EDTA, and 255 mM sucrose, pH 7.4. Store at 2-8 C. 5. Aprotinin, stock solution 10 mg mL in double-distilled (dd) H2O. Store at 20 to 5 C for up to 6 mo. 6. Leupeptin, stock solution 5 mg mL in ddH2O. Store at 20 to 5 C for up to 6...

Introduction

The study of transcriptional processes in higher eukaryotes has been limited by the scarce availability of in vivo assays. This shortage of technical approaches has become more important in light of the emerging notion that the natural chromatin context affects the outcome of transcriptional activation in vivo (1). Chromatin can exert a regulatory effect on transcription by modulating the access of activators to DNA (1). Different posttranslational modifications of specific residues in the...

Materials Reagents

Dulbeco's modified Eagle's medium (DMEM) containing 10 calf serum is used to culture 3T3-L1 cells. Inositol-free DMEM containing myo-(2-3H)-inositol and 10 calf serum should be used for labeling cellular phosphoinositides. DMEM, inositol-free DMEM, and calf serum are products of Gibco (Hyclone, Logan, UT). 2. HPLC-grade acetic acid, n-butanol, chloroform, ethanol, methanol, pyridine, petroleum ether, diethyl ether, and ethyl formate can be obtained from Fisher Scientific (Pittsburgh, PA)....

Methods

Split insulin-producing cells into eight-well chamber slides and allow to grow until they are approx 50 confluent (see Note 1). 2. Wash cells four times in 1X PBS. 3. Fix cells by adding a large volume of ice-cold methanol and incubating at 4 C for 10 min (see Note 2). 4. Remove methanol and wash once in 1X PBS. 5. Add 200 iL blocking buffer and incubate at room temperature for 15 min (see Note 3). 6. Discard blocking buffer and add insulin primary antibody, which has been diluted 1 400 in 200...

Purification of Single Cells and Nonft Cells

The dispersed islet cells are washed in isolation medium containing 2.8 mM glucose and submitted to auto-fluorescence-activated cell sorting (FACS) using a FAC- Fig. 1. FACS analysis of unpurified islets cells examined for their FAD fluorescence and FSC intensity at 2.8 mM glucose. The subpopulation with high FAD and high FSC represents the b-cells, whereas the islet non b-cells are lower in FAD content and cause less FSC. Fig. 1. FACS analysis of unpurified islets cells examined for their FAD...

Reverse Transcriptase Polymerase Chain Reaction

All reagents for both the reverse transcription and PCR used in this protocol are widely available from a number of commercial sources (e.g., Promega). 5' Primer (20 pmol L) 5' GCGGGCTGCGTCTAGTTGCAGTAG-3' 3' primer (20 pmol L) 5' ATGGCCCTGTGGATGCGCCTCCTG-3'. 7. Reverse transcriptase (Moloney murine leukemia virus, MMLV-RT). 10. Stock solutions of NTPs and dNTPs. 11. 5X First-strand buffer (for reverse transcription).

TL Preadipocyte Adipocyte Tissue Culture

3T3-L1 preadipocytes or adipocytes should be grown to at least 60 confluency in 75-cm2 flasks (6 mL of medium). 2. The medium (DMEM +10 calf serum) should be decanted from flasks and cells should be washed with fresh inositol-free DMEM +10 calf serum and cultured in Fig. 1. Scheme for the determination of phosphoinositide concentrations in 3T3-L1 cells. Actively dividing 3T3-L1 cells are metabolically labeled, then, total lipids are extracted and deacylated and analyzed using strong anion,...

Transfection

Cell culture medium containing 2X serum concentration Dulbecco's modified Eagle's medium (DMEM) plus 20 fetal bovine serum (FBS) (Sigma or Hyclone). 2. Lipofectamine reagent (Invitrogen) or other commercially available products. 3. Optimem I Reduced Serum Medium (Invitrogen). 4. Phosphate-buffered saline (PBS) 150 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4. 5. Plasmid DNA-reporter gene construct containing the full length, or a part, of the insulin gene promoter.

Incubation of Isolated Muscles

Epitrochlearis muscles are removed under anesthesia from rats weighing approx 100 g (5,8). These muscles are ideally suited for incubation because they are thin and flat and give good glucose-transport responses to contraction (9). Alternatively, soleus muscles can be used for these experiments however, these muscles do not respond as well to contractions and must be split prior to use in incubations, due to their thickness (8,10). 2. The excised muscles are rinsed in 0.9 saline and placed in 2...

Amplification of the GFP Adenovirus in HEK Cells

Tissue culture dishes of 6 cm, 10 cm, and 15 cm diameter and sterile cell scrapers 15-mL and 50-mL conical tubes. 2. Growth medium for HEK 293 cells minimal essential medium (MEM) containing 5 fetal bovine serum (FBS) (Sigma), 2 mM glutamine, 50 iM strepomycin peni-cillin was sterilized through a 0.2- m filter. 3. 0.05 Trypsin-0.53 mM EDTA 4Na. 4. 1X Phosphate-buffered saline (PBS) 150 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4 (pH 7.4) sterilize through a 0.2- m filter and store at 4...

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...

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...

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...