Isolation of Dense Core Secretory Vesicles from Pancreatic Endocrine Cells by Differential and Density Gradient Centrifugation

John C. Hutton1, Randall Wong1, Howard W. Davidson1

1 Barbara Davis Center for Childhood Diabetes, University of Colorado at Denver and Health Sciences Center, Aurora, Colorado
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.32
DOI:  10.1002/0471143030.cb0332s42
Online Posting Date:  March, 2009
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Abstract

Methods are presented for the separation of dense core secretory vesicles from insulin‐secreting tissues (insulin granules) based on a combination of differential and density gradient centrifugation on various media. Emphasis is given to the use of transplantable tumors, tissue culture cell lines, and pancreatic islets as a tissue source. Curr. Protoc. Cell Biol. 42:3.32.1‐3.32.20. © 2009 by John Wiley & Sons, Inc.

Keywords: insulin granules; DCV; insulinoma; Min6; INS1; homogenization; centrifugation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of DCVs from Insulinomas
  • Preparation of Granules on Preformed Nycodenz Gradients
  • Alternate Protocol 1: Preparation of Granules Using Nycodenz Continuous Gradients
  • Alternate Protocol 2: Using Nycodenz Step Gradients for Crude Granule Preparations
  • Alternate Protocol 3: Granule Purification by Combined Nycodenz‐Percoll Gradients
  • Alternate Protocol 4: Separations Using Self‐Forming Nycodenz Gradients
  • Insulin Granule Preparations from other Tissue Sources
  • Basic Protocol 2: Preparation of Granules from Cultured Min6 Insulinoma Cells
  • Support Protocol 1: Propagation of Solid Tumors
  • Enzyme Marker Assays
  • Support Protocol 2: Enzyme Assay for β,N‐Acetyl Glucosaminidase
  • Support Protocol 3: Enzyme Assay for NADPH–Cytochrome c Reductase
  • Support Protocol 4: Enzyme Assay for Cytochrome Oxidase
  • Support Protocol 5: Enzyme Assay for Carboxypeptidase E
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of DCVs from Insulinomas

  Materials
  • Rats (NEDH strain, either sex, 150 to 200 g)
  • 70% (v/v) ethanol or iodine
  • Sterile phosphate‐buffered saline: 10 mM Na phosphate in 150 mM NaCl, pH 7.4, on ice
  • Ice‐cold β‐cell homogenization medium (β‐HM; see recipe)
  • 27% Percoll medium (see recipe)
  • Sterile dissection instruments (large and small scissors, large and small forceps, scalpels)
  • Sterile Petri dishes
  • 50‐ml sterile plastic centrifuge tubes
  • Refrigerated low‐speed centrifuge accepting 50‐ml capacity tubes (2.5‐cm diameter)
  • Dounce homogenizer (40‐ml capacity with loose (B) and tight (A) glass pestles; Wheaton)
  • Cheesecloth
  • Potter‐Elvehjem homogenizer (25‐ml capacity, Teflon pestle, 0.1‐mm nominal clearance—a bench or wall‐mounted variable speed drive capable of 2500 rpm for homogenization or a domestic drill mounted in a drill press)
  • 9 × 2.5–cm polycarbonate centrifuge tubes
  • 30‐ml syringe fitted with lumbar puncture needle or peristaltic pump
  • High‐speed refrigerated centrifuge with Beckman Type 30 and SW28 rotors

Alternate Protocol 1: Preparation of Granules Using Nycodenz Continuous Gradients

  Materials
  • β‐HM medium (see recipe)
  • Nycodenz
  • Post‐nuclear supernatant (PNS; see protocol 1)
  • 9 × 2.5–cm polycarbonate centrifuge tubes
  • Parafilm
  • Multichannel peristaltic pump (Fig. )
  • Beakers
  • Stir plate and magnetic stir bar
  • Velcro
  • Pasteur pipets with 0.5‐cm wide bores
  • Centrifuge with a Beckman SW28
  • 5‐ml syringe with long (8‐cm) needle, 16‐G lumbar puncture set

Alternate Protocol 2: Using Nycodenz Step Gradients for Crude Granule Preparations

  Materials
  • Post‐nuclear supernatant (PNS; see protocol 1)
  • 4.4%, 8.8%, and 17.2% (v/v) Nycodenz solutions (see recipes)
  • β‐HM medium (see recipe)
  • 9 × 2.5–cm polypropylene centrifuge tubes
  • Centrifuge with a Beckman SW28 rotor
  • 5‐ml syringe with 16‐G needle

Alternate Protocol 3: Granule Purification by Combined Nycodenz‐Percoll Gradients

  Materials
  • Nycodenz step gradient (see protocol 3)
  • 27% (v/v) Percoll (see recipe)
  • β‐HM medium (see recipe)
  • 9 × 2.5–cm polycarbonate centrifuge tube
  • Refrigerated centrifuge with Beckman Type 30 and SW28 rotors

Alternate Protocol 4: Separations Using Self‐Forming Nycodenz Gradients

  Materials
  • 27.6% isotonic Nycodenz
  • β‐HM (see recipe)
  • Post‐nuclear supernatant (PNS; see protocol 1)
  • 13 × 50–mm centrifuge tubes
  • Refrigerated ultracentrifuge with a Beckman SW50.1 swing‐out rotor

Basic Protocol 2: Preparation of Granules from Cultured Min6 Insulinoma Cells

  Materials
  • 50× media supplement stock (see recipe)
  • Basal RPMI 1640 medium (see recipe)
  • Mm 6 cells in culture (75% to 80% confluent in 75‐cm2 tissue culture flask; Miyazaki)
  • 0.25% (w/v) trypsin‐EDTA (GIBCO cat. no. 25200)
  • Calcium‐ and magnesium‐ free PBS (GIBCO cat. no. 10010)
  • Cell dissociation medium (GIBCO cat. no. 13150‐016)
  • β‐HM (see recipe)
  • DNase I
  • 30% Percoll (see recipe)
  • 150‐cm2 tissue culture flasks
  • 150‐mm Petri dishes treated for tissue culture (Nunc cat. no. 168381)
  • Cell lifter (Costar cat. no. 3008)
  • 2‐ml Dounce homogenizer with loose‐fitting (A) and tight‐fitting (B) glass pestles (Wheaton), pre‐chilled
  • 1.5‐ml microcentrifuge tubes
  • 70‐µm mesh filter or insulin syringe and 28‐G needle (Becton Dickenson cat. no. 309309)
  • 3‐ml syringe
  • Ball‐bearing cell cracker (0.1576‐in. ball; 0.1586‐in. bore; Industrial Tectonics)
  • 13 × 56–mm polycarbonate centrifuge tubes
  • Centrifuge with Beckman TLA110 rotor
  • Screw‐capped microcentrifuge tubes

Support Protocol 1: Propagation of Solid Tumors

  Materials
  • Ethanol
  • Tumor pieces in PBS (see protocol 1) in 1‐ml syringe and 16‐ or 18‐G needle

Support Protocol 2: Enzyme Assay for β,N‐Acetyl Glucosaminidase

  Materials
  • Sample material: gradient fractions
  • β,N‐acetyl glucosaminidase reaction mixture (see recipe)
  • 1 M Na 2CO 3
  • 4‐Methylumbelliferone (standard)
  • 96‐well plate suitable for fluorimeter
  • 37°C heating block
  • Fluorimeter

Support Protocol 3: Enzyme Assay for NADPH–Cytochrome c Reductase

  Materials
  • Samples
  • NADPH‐cytochrome c reductase reaction mixture (see recipe)
  • 96‐well plate
  • Spectrophotometer

Support Protocol 4: Enzyme Assay for Cytochrome Oxidase

  Materials
  • Samples
  • Cytochrome oxidase reaction mixture (see recipe)
  • 100 mM potassium ferricyanide
  • 96‐well plate
  • Spectrophotometer

Support Protocol 5: Enzyme Assay for Carboxypeptidase E

  Materials
  • Samples: granules isolated from insulinomas or cell lines
  • Carboxypeptidase E reaction mix A (see recipe)
  • Carboxypeptidase E reaction mix B (see recipe)
  • Acidified chloroform (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • 37°C heating block
  • 96‐well silica glass plate
  • Fluorimeter (360‐nm excitation; 500‐nm emission)
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Figures

Videos

Literature Cited

Literature Cited
   Asfari, M., Janjic, D., Meda, P., Li, G., Halban, P.A., and Wollheim, C.B. 1992. Establishment of 2‐mercaptoethanol‐dependent differentiated insulin‐secreting cell lines. Endocrinology 130:167‐178.
   Bergman, B., McManaman, J.L., and Haskins, K. 2000. Biochemical characterization of a beta cell membrane fraction antigenic for autoreactive T cell clones. J. Autoimmun. 14:343‐351.
   Bhathena, S.J., Oie, H.K., Gazdar, A.F., Voyles, N.R., Wilkins, S.D., and Recant, L. 1982. Insulin, glucagon, and somatostatin receptors on cultured cells and clones from rat islet cell tumor. Diabetes 31:521‐531.
   Chick, W.L., Warren, S., Chute, R.N., Like, A.A., Lauris, V., and Kitchen, K.C. 1977. A transplantable insulinoma in the rat. Proc. Natl. Acad. Sci. U.S.A. 74:628‐632.
   Efrat, S., Linde, S., Kofot, H., Spector, D., Delannoy, M., Grant, S., Hanahan, B., and Baekeskov, S. 1988. Beta‐cell lines derived from transgenic mice expressing a hybrid insulin gene‐oncogene. Proc. Natl. Acad. Sci. U.S.A. 85:9037‐9041.
   Hutton, J.C. 1982. The internal pH and membrane potential of the insulin‐secretory granule. Biochem. J. 204:171‐178.
   Hutton, J.C. and Peshavaria, M. 1983. Nucleotide and bivalent cation specificity of the insulin‐granule proton translocase. Biochem. J. 210:235‐242.
   Hutton, J.C., Penn, E.J., and Peshavaria, M. 1982. Isolation and characterisation of insulin secretory granules from a rat islet cell tumour. Diabetologia 23:365‐373.
   Hutton, J.C., Peshavaria, M., and Tooke, N.E. 1983. 5‐Hydroxytryptamine transport in cells and secretory granules from a transplantable rat insulinoma. Biochem. J. 210:803‐810.
   Konrad, R.J., Young, R.A., Record, R.D., Smith, R.M., Butkerait, P., Manning, D., Jarett, L., and Wolf, B.A. 1995. The heterotrimeric G‐protein Gi is localized to the insulin secretory granules of beta‐cells and is involved in insulin exocytosis. J. Biol. Chem. 270:12869‐12876.
   Madsen, O.D., Larsson, L.I., Rehfeld, J.F., Schwartz, T.W., Lernmark, A., Labrecque, A.D., and Steiner, D.F. 1986. Cloned cell lines from a transplantable islet cell tumor are heterogeneous and express cholecystokinin in addition to islet hormones. J. Cell Biol. 103:2025‐2034.
   Miyazaki, J., Araki, K., Yamato, A., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., and Yamamura, K. 1990. Establishment of a pancreatic beta cell line that retains glucose‐inducible insulin secretion: Special reference to expression of glucose transporter isoforms. Endocrinology 127:126‐132.
   Mohlig, M., Wolter, S., Mayer, P., Lang, J., Osterhoff, M., Horn, P.A., Schatz, H., and Pfeiffer, A. 1997. Insulinoma cells contain an isoform of Ca2+/calmodulin‐dependent protein kinase II delta associated with insulin secretion vesicles. Endocrinology 138:2577‐2584.
   Praz, G.A., Halban, P.A., Wollheim, C.B., Blondel, B., Straus, A.J., and Renold, A.E. 1983. Regulation of immunoreactive‐insulin release from a rat cell line (RINm5F). Biochem. J. 210:345‐352.
   Roep, B.O., Kallan, A.A., Hazenbos, W.L., Bruining, G.J., Baileys, E.M., Arden, S.D., Hutton, J.C., and de Vries, R.R. 1991. T‐cell reactivity to 38‐kD insulin‐secretory‐granule protein in patients with recent‐onset type 1 diabetes. Lancet. 337:1439‐1441.
   Sottocasa, G.L., Kuylenstierna, B., Ernster, L., and Bergstrand, A. 1967. An electron‐transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol. 32:415‐438.
   Wang, S.Y. 1989. The acute effects of glucose on the insulin biosynthetic‐secretory pathway in a simian virus 40‐transformed hamster pancreatic islet beta‐cell line. Endocrinology 124:1980‐1987.
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