Association of AMP-activated Protein Kinase Subunits With Glycogen Particles as Revealed In Situ by Immuno-electron Microscopy

Moise Bendayan ^1*, Irene Londono ¹, Bruce Kemp ¹, Grahame D. Hardie ¹, Neil Ruderman ¹ and Marc Prentki ¹

¹ Department of Pathology and Cell Biology (MB,IL) and Departments of Nutrition and Biochemistry (MP), Montreal Diabetes Center, University of Montreal, Montreal, Quebec, Canada; St. Vincent's Institute of Medical Research, University of Melbourne, Fitzroy, Victoria, Australia (BK); Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom (GDH); and Diabetes Research Unit, University of Boston, Boston, Massachusetts (NR)

^* To whom correspondence should be addressed. E-mail: moise.bendayan{at}umontreal.ca.

Submitted on March 25, 2009
Accepted on 18 June 2009

	Abstract

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-protein kinase subunits in liver tissue of normal rats fed ad libitum. AMPK and subunits were located both in the cytosol and in close association with rosettes of glycogen particles (-particles). In order to reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by post-fixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the 1 and 2 subunits of AMPK was found to be about twofold greater over glycogen than over cytosol, whereas labeling for 1 was 8 fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the 1 subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches.

Key Words: AMP-activated kinase, glycogen, immunocytochemistry, protein A-gold, liver tissue

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

Guidelines | Subscriptions | About | exPRESS - Current - Archive | Business Information | Contact

The Journal of Histochemistry & Cytochemistry is owned, published, and licensed by The Histochemical Society © 2009