In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker

Abstract

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.

Figure 1.

Overexpression of GFP-LC3 does not affect the autophagic status of F9 cells. (A) F9 cells overexpressing GFP-LC3 were incubated in Hanks' solution for the indicated time and directly observed by fluorescence microscopy. Bar, 10 μm. (B) Total cell lysates were prepared from wild-type F9 cells and F9 cells overexpressing GFP-LC3 before or after 2-h amino acid starvation and analyzed by immunoblotting using anti-LC3 antibody. The band intensities of endogenous LC3-I and LC3-II were quantified. The LC3-II/LC3–1 ratio was shown. (C) Wild-type F9 cells and F9 cells overexpressing GFP-LC3 before or after 2-h amino acid starvation were examined by electron microscopy and morphometric analysis. The ratio of the total area of autophagic vacuoles to the total cytoplasmic area is shown.

Figure 2.

Expression of GFP-LC3 in the GFP-LC3 transgenic mice. Twenty-five milligrams of each tissue homogenate was subjected to SDS-PAGE and analyzed by immunoblotting with anti-GFP and anti-LC3 antibodies.

Figure 3.

Liver autophagy in response to starvation. (A–C) Liver samples were prepared from GFP-LC3 transgenic mice before (A) or after 24-h starvation (B and C) and fixed with 4% paraformaldehyde. Cryosections were analyzed by fluorescence microscopy. Panel C demonstrates the most highly induced case. Bar, 10 μm. (D and E) Localization of GFP-LC3 in hepatocytes from 24-h starved GFP-LC3 mice. Liver samples were prepared from GFP-LC3 transgenic mice after 24-h starvation and fixed with 4% paraformaldehyde. The localization of GFP-LC3 was examined by silver-enhanced immunogold electron microscopy using an anti-GFP antibody. A cup-shaped isolation membrane (arrow in D) and double-membrane autophagosome (arrow in E) were shown. Bar, 1 μm.

Figure 7.

Quantitative analysis of the formation of GFP-LC3 dots during starvation. The number of GFP-LC3 dots was counted and divided by the corresponding area. For analysis of skeletal muscles, transverse sections were used. Because the glomerulus and thymic cortex consist of several kinds of cells, the actual densities of GFP-LC3 dots in podocytes and thymic epithelial cells were not calculated. The Y-axis indicates the number of GFP-LC3 dots (×10⁴/mm²). Each value represents the mean ± SD of at least five mice. ^*p < 0.05 and ^**p < 0.01.

Figure 4.

Autophagy in muscle tissues. (A and B) GFP images of transverse sections of extensor digitorum longus (EDL) muscles (A) and soleus muscles (B) after the indicated starvation periods. Bar, 10 μm. (C and D) Longitudinal section of EDL muscles after 24-h starvation (C) and soleus muscles after 48-h starvation (D). Asterisks indicate nuclei. Bar, 10 μm. (E) GFP images of heart muscles at 0-, 24-, and 48-h starvation. Bar, 10 μm. (F and G) Immunoelectron microscopic analysis of gastrocnemius muscle after 24-h starvation. Autophagosomes associated with GFP-LC3 (arrows) accumulate between myofibrils (F) and at perinuclear regions (G). Bar, 1 μm. (H) Immunoelectron microscopic analysis of heart muscles from 24-h starved mice. A cluster of autophagosomes is generated between myofibrils (arrows). The double arrow indicates an autophagosome enclosing a mitochondrion. Bar, 1 μm.

Figure 5.

Autophagy in pancreatic exocrine cells. (A) GFP images (top panels) and corresponding differential interference contrast (DIC) images (bottom panels) at the indicated starvation periods are shown. Bars, 10 μm. (B) Immunoelectron microscopic analysis of pancreatic acinar cells. Before starvation, large zymogen granules and small autophagic vacuoles (arrows) are observed (left panel). After 24-h starvation, the size of autophagosomes is enlarged, and some zymogen granules are observed in the autophagosomes (double arrows; right panel). Bar, 1 μm. (C) Amounts of pancreatic proteases decrease during starvation. Homogenates were prepared from three independent mice before and after 24-h starvation and subjected to immunoblot analysis using antitrypsin, antiamylase, antielastase, and anticarboxypeptidase antibodies. Anti-tubulin antibody was used as a control.

Figure 6.

Tissues showing high levels of autophagy before starvation. (A) GFP fluorescence of the glomerulus from a fed mouse. Bar, 10 μm. Inset: high-magnification images from other areas. Bar, 2 μm. (B) Immunoelectron microscopy of a podocyte using anti-GFP antibody. The arrows indicate autophagosomes. Bar, 1 μm. (C) The thymus cortex from a fed mouse. GFP fluorescence, anticytokeratin staining, and a merged image are shown. Bar, 10 μm. (D) Immunoelectron microscopy of the thymic cortex. The arrow indicates an autophagosome in a stromal epithelial cell. Bar, 1 μm. (E) A GFP image of the lens epithelial cells of a fed mouse. Bar, 10 μm. Insets: high-magnification images from other sections. Some GFP-LC3–positive structures in epithelial cells are detected as ring- and cup-shaped. Bar, 1 μm. (F) Immunoelectron microscopy of the lens. An arrow and asterisk indicate the autophagosome and a lens fiber, respectively. Bar, 1 μm.

Figure 9.

Distinction between GFP-LC3 and autofluorescence signals. Samples from the frontal cortex of the brain (A) and the medulla of the thymus (B) were analyzed for green (left panels) and red (middle panels) fluorescence. Merged images are shown in the right panels. GFP-specific signals (arrows) and autofluorescent signals (arrowheads) are indicated. A neuron-like cell (A) and stromal cells show autofluorescence. Similar autofluorescent signals were also observed in samples from nontransgenic mice. Bar, 10 μm.

Figure 8.

Conversion of LC3-I to LC3-II in mouse tissues during 24-h starvation. Tissue homogenates were prepared from three fed mice and three 24-h starved mice and subjected to immunoblot analysis using anti-LC3 antibody. The positions of LC3-I and LC3-II are indicated.