Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy

Abstract

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK alpha and beta subunits were located both in the cytosol and in close association with rosettes of glycogen particles (alpha particles). 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 postfixation 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 alpha(1) and alpha(2) subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for beta(1) was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the beta(1) subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches.

Figure 1

Results from Western blot analyses using two of the antibodies against the AMP-activated protein kinase (AMPK) β₁ subunit. The antibody from Santa Cruz (A) and that from Epitomics (B,C) were tested against liver tissue from normal rat (RAT), normal wild-type mouse (MWT), and AMPK β₁ knockout mouse (MKO). A single band between 37 and 40 kDa was obtained with tissues from normal animals, whereas no band was detected with liver tissue from the knockout mouse.

Figure 2

Liver tissue from normal rats fed at libitum. Protein A–gold immunocytochemistry for the AMPK subunits. Tissue sample fixed with glutaraldehyde and embedded in Lowicryl. The glycogen deposits have been extracted, leaving empty spaces within the cytoplasm (asterisks). Other structures such as mitochondria (M) and rough endoplasmic reticulum (RER) are well preserved. The gold particles revealing the AMPK α₁ subunit are present in the cytosol associated with the RER. They are absent over the clear areas where the glycogen deposits have been extracted. Bar = 1 μm.

Figure 3

Liver tissue from normal rats fed at libitum. Protein A–gold immunocytochemistry for the AMPK subunits. Liver sample fixed with the paraformaldehyde–glutaraldehyde followed by postfixation with the osmium tetroxide–lead citrate combination and embedded in Epon. The glycogen deposits (Gly) are retained as electron-dense particles arranged in rosettes. They are labeled for the AMPK subunit α₁. The gold particles are preferentially associated with glycogen deposits. Mitochondria (M) display very few particles. Bar = 1 μm.

Figure 4

Liver tissue from normal rats fed at libitum. Protein A–gold immunocytochemistry for the AMPK subunits. Liver sample fixed with the paraformaldehyde–glutaraldehyde followed by postfixation with the osmium tetroxide–lead citrate combination and embedded in Epon. The glycogen deposits (Gly) are retained as electron-dense particles arranged in rosettes. They are labeled for the AMPK subunit β₁. The gold particles are preferentially associated with glycogen deposits. Some particles are also present over the cytosol associated with the rough endoplasmic reticulum (RER) whereas mitochondria (M) display very few particles. Bar = 1 μm.

Figure 5

Liver tissue from normal rat fed at libitum. Protein A–gold immunocytochemistry for AMPK-β₁ subunit. At very high magnification, we can assess the position of the gold particles (AMPK β₁) over the glycogen rosettes. The gold particles are mostly at a peripheral location on the glycogen rosettes. Bar = 0.1 μm.

Figure 6

Liver tissue from normal rat fed at libitum. Protein A–gold immunocytochemistry for AMPK-β₁ subunit. Histogram revealing the distribution of the gold particles (AMPK β₁) over the glycogen rosettes. The evaluation was carried out in the following way: the distance (d) between the gold particle and the center of the corresponding rosette was measured and divided by the radius (r) of the same glycogen rosette. The ratio (R) between these two values gives a good assessment of the position of the gold particle in reference to the glycogen rosette. The histogram represents the number of gold particles according to R (ratio) values. A value of 1 indicates that the gold particle is exactly at the edge of the glycogen rosette. The average value obtained was 0.96 ± 0.02, which indicates that the very large majority of the particles are located at the edge of the glycogen rosette. Some particles are located slightly outside the glycogen rosettes (R value >1); this is due to the presence of protein layers (protein A and IgGs) around the gold particles, which then appear to be slightly away from the glycogen rosette.