Induction of Autophagy by Amino Acid Starvation in Fish Cells

Takeshi Yabu Shintaro Imamura Nanami Mizusawa Ken Touhata Michiaki Yamashita 0 ) National Research Institute of Fisheries Science , 2-12-4 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-8648, Japan Autophagy is well established as a starvationinduced process in yeast and mammalian cells and tissues. To elucidate the cellular mechanisms induced by starvation in fish, we characterized the induction of autophagy in cultured zebrafish cells under starvation conditions. As an autophagic marker protein, the microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) was cloned from the fish cells, and its expression and localization were characterized. In zebrafish embryonic (ZE) cells, posttranslational modifications produced two distinct forms of MAP1-LC3B, i.e., a cytosolic form and a membranebound form (types I and II, respectively). Immunofluorescence microscopy revealed fluorescently labeled autophagosomes in cells stably transfected with a green fluorescent protein (GFP)-MAP1-LC3B fusion protein and showed that this protein accumulated in punctate dots in a timedependent manner in response to amino acid starvation. Starvation also induced the degradation of long-lived proteins. Treatment with 3-methyladenine and wortmannin, two class-III inhibitors of phosphoinositide 3-kinase (PI3K), repressed autophagy under starvation conditions, indicating that the PI3K class-III pathway regulates starvation-induced autophagy in fish. - Many species of fishes undergo natural periods of starvation attributable to spawning migration activity or seasonal changes in food supply, and some can survive without feeding for several months or years (Lin et al. 1977; Moon and Johnston 1979). Numerous studies on the effects of starvation on fish tissues and muscle and hepatic enzymes have shown that the metabolic response to starvation includes the induction of proteolytic enzyme activities and energy consumption (Larsson and Lewander 1973; Lin et al. 1977; Moon and Johnston 1979; Patterson et al. 1974). Spawning migration of salmon, which is apparently supported by protein catabolism, is accompanied by the depletion of carbon and energy sources from muscle and liver and the stimulation of gluconeogenesis from amino acids (French et al. 1983). In carp, proteins and particular amino acids appear to be favored over carbohydrates as energy sources (Shimeno et al. 1990). Studies focusing on the autophagosomallysosomal pathway in rainbow trout recognized that this pathway is regulated during muscle wasting (Mommsen 2004; Salem et al. 2006). Thus, protein degradation by autophagy is an important adaptive mechanism in fish that allows them to survive nutritional starvation. When autophagy is induced by nutrient starvation or other stresses or chemical agents, autophagosomes envelop cytoplasmic constituents, including organelles (Cuervo 2004). The double membrane-bound autophagosome subsequently fuses with lysosomes to become an autolysosome; within this structure, lysosomal hydrolytic enzymes degrade the cytoplasm-derived material. The most important marker protein for detecting autophagic induction is microtubule-associated protein 1-light chain 3B protein (MAP1-LC3B) (Ichimura et al. 2000; Kirisako et al. 2000; Lang et al. 1998; Schlumpberger et al. 1997; Tanida et al. 1999). MAP1-LC3 exists in two modified forms: an 18-kDa cytoplasmic form that was originally identified as a MAP1-LC3 subunit (Kuznetsov and Gelfand 1987; Mann and Hammarback 1994) and a 16-kDa form that is associated with autophagosome membranes (Kabeya et al. 2000). The latter form is generated through a multistep posttranslational modification process that includes proteolytic cleavage of the C-terminal residue (Gly-120) from the newly synthesized MAP1-LC3 protein (Kabeya et al. 2000). In cultured cells and transgenic animals, autophagosomes can be fluorescently labeled in vivo by the transduction and expression of a chimeric protein consisting of green fluorescent protein (GFP) fused to MAP1-LC3B (Mizushima et al. 2004). The genes related to autophagy have been isolated in zebrafish, and the expression of GFPMAP1-LC3B expression has been reported (He et al. 2009; He and Klionsky 2010; Makky et al. 2007). More recently, muscle protein degeneration in rainbow trout in vivo and in vitro was investigated in several autophagy-related genes, i.e., LC3B, gabarapl1, atg12l, and atg4b (Seiliez et al. 2010). Fasting fish for 14 days or serum depletion of trout myocytes strongly induced the expression of all studied genes. Insulin-like growth factor-1 (IGF1) induced FoxO3 phosphorylation in an in vitro primary culture of rainbow trout muscle cells but no effect on the expression of autophagyrelated genes (Seiliez et al. 2010). Schitz et al. (2010) used electron microscopy to observe the induction of double membrane autophagosomes in cultured Atlantic salmon cells infected with infectious salmon anemia virus. Our preceding studies observed macroautophagy and chaperonemediated autophagy with the expression of MAP1-LC3B and HSC70 expression as biomarkers in cells for the fish cultured cells derived from zebrafish and yellowtail under heat-shock conditions by Western blotting and immunocytochemistry (Yabu and Yamashita 2008; Yabu et al. 2011; Yamashita 2010). These previous studies indicate that MAP1-LC3 genes can be isolated and establish the fish models for autophagy and starvation studies. Thus, the GFPMAP1-LC3B fusion protein can be used as an important biomarker to visualize fluorescent autophagosomes in fish cells in response to starvation. In this study, we examined the autophagic response to starvation in fish using MAP1-LC3B as a marker for autophagosome formation. By searching the zebrafish Expressed Sequence Tag (EST) DNA database in GenBank, we identified three distinct zebrafish homologs (, , and ; MAP1-LC3A, MAP1-LC3B, and MAP1-LC3C, respectively) of rat MAP1-LC3. We cloned these zebrafish proteins and also cloned MAP1-LC3B homologs from three other fish species: bluefin tuna (Thunnus orientalis), yellowtail (Seriola quinqueradiata), and Japanese flounder (Paralichthys olivaceus). We generated stable zebrafish embryonic (ZE) cell lines transfected with a GFPMAP1-LC3B fusion gene construct and examined protein degradation by autophagy in these cells under conditions of nutritional stress. Treatment with inhibitors of phosphoinositide 3-kinase (PI3K) repressed starvation-induced autophagy, allowing us to identify the major autophagic pathway operating in cultured fish cells and tissues under starvation conditions. Materials and Methods Wortmannin, 3-methyladenine (3-MA), mouse anti-tubulin monoclonal antibody (T6074), and mouse anti-bovine HSC70 IgG (BRM-22) were purchased from SigmaAldrich (St. Louis, MO, USA). Cell culture reagents, ThermoScript RT-PCR System, and Xpress System synthetic oligonucleotides were obtained from Invitrogen (Carlsbad, CA, USA). A monoclonal antibody against enhanced GFP, the mammalian expr (...truncated)