The Function Of Endoplasmic Reticulum Stress Signaling Protein Xbp1in Starved Drosophila And The Possible Mechanism

Endoplasmic reticulum (ER) exists in all the eukaryotic cells, which is the essential organelle that controls the synthesis, modification, processing and transporting of secreting and transmembrane proteins. When the newly synthesized peptides exceed the capability of the ER to process or the cell encounters a unfavorable condition such as Ca2+ loss from ER to cytosol, the unfolded or misfolded proteins will accumulate in the ER lumen, therefore, the ER will be forced to a stressful state, which is defined as ER stress. Once ER-stress occurs, the ER stress sensors on the ER membrane will activate a series of signaling pathways called unfolded protein response (UPR) to enhance protein processing capacity in ER or to reduce the synthesis of proteins for ER to process and finally to attenuate stress. IRE1-XBP1 signaling pathway is the most conservative one in UPR reaction. Some previous data in our group showed that, in mouse liver, the IRE1-XBP1 signaling pathway is activated under starvation condition, and it regulates the metabolism adaptation response incurred by starvation via modulating the expression of (PPAR α (proliferation activated receptor alpha). In this thesis, we exploited drosophila as model animals, and focused the experiments on its fat body, which is the organ closely related to energy storage and metabolism. Our results indicated that the ER stress protein XBP1 in drosophila (dXBP1) is able to reduce the consumption of fat, and extends the life span of Drosophila under starvation condition via enhancing the degradation of Forkhead box O protein in drosophila (dFOXO). Our results also showed that the process of degradation is proteasome-dependent rather than insulin- signaling-pathway dependent. In summary, our data indicate that ER stress may be involved in the starvation response and regulation of fat mobilization under starvation condition. The research has theoretical and practical significance.Aim:To explore the regulatory function of endoplasmic reticulum stress protein XBP1 on fat metabolism in drosophila under starvation condition and the possible mechanisms.Methods:1. The control group, dXbp1 knockout group, dXbp1s over expression group of drosophila and 3 instar larvae were treated with normal feeding and starvation (drosophila 48 hours and larvae 8 hours). The fat body tissues of drosophila and larvae were stained with Nile Red and the consumption of fat in fat body was observed. The triglyceride level and glycogen level in drosophila after 48 hours starvation were determinated.2. The control group, dXbp1 knockout group and dXbp1s over expression group of drosophila were food-starved continuously. The numbers of dead drosophila per 12 hours were calculated and the survival rate curve of drosophila with log-rank in Graphpadsoftware was applied to explore the lifespan of various genotype on the drosophila under starvation conditions.3. The mRNA levels of genes related to fat accumulation (FoxO, Bmm,4ebp, InR, Fas, Hsl) in different genotype under normal feeding and starvation conditions were determinated by qPCR, and the possible mechanism was analyzed.4. Immunofluorescence staining and Western Blotting were exploited to further explore the mechanism of endoplasmic reticulum stress protein XBP1 in the regulation of Drosophila starvation response.5. Morphological observation techniques were applied to explore the effects of abnormal expression of dXBP1 anddFOXO on the lipid droplet accumulation and on the development of drosophila larvae.Results:1. In the dXbp1 knockout group, the lipid droplet content in the drosophila and the 3 instar larvae under starvation condition was significantly lower than that in the normal feeding group. The triglyceride level of the Drosophila in dXbp1 knockout group under starvation condition were significantly lower than in the normal feeding condition.2. The results of survival rates of Drosophila under continuous starvation condition showed, all the drosophila in the dXbpl knock down group were dead at 4.5 day, and the half life time (time point at which half flies were dead) was significantly different compared to that of control group with normal feeding. All the drosophila in the dXbp1 over expression group died at 9.5 day, and the half life time was significantly different compared to that of control group with normal feeding.3. The qPCR results exhibited that the mRNA levels of dFoxO in drosophila from both dXbp1 knock down group and dXBPl over expression group were not significantlychanged. However, the mRNA levels of dFoxO downstream genes were significantly changed, such as the mRNA level of Bmm in dXbpl knock down group was significantly increased under starvation condition, which was different from that of normal feeding control. The mRNA level of Hsl had a similar variation trend in different dXbpl expression group between normal feeding control and starvation experimental group.4. Immunofluorescence staining of fat body cells in Drosophila showed dFOXO protein level was obviously decreased in the cytoplasm of the fat body cell compared to the normal feeding control group. dFOXO was significantly decreased when co-over expression with dXBP1s. Mcd8 was used as a control did not showed obvious change.5. Western blotting results showed, the level of dFOXO protein decreased with the increase of dXBPls expression, and both molecules were negatively correlated; After CHX treatment, dFOXO protein was more stable than dXBP1s protein. And over-expression dXBPls still significantly reduced the level of dFOXO protein; In the absence of MG132 treatment, over expression of dXBP1s significantly reduced the level of dfOXO protein, and the expression level of dXBPls was not significantly changed. In dXBP1 and dFOXO co-over expression group, the protein level of dFOXO was significantly higher upon treatment with MG132 than that of the non-MG132 treatment group. dFOXO degradation caused by overexpression of dXBP1s was not obvious when treated with MG132; Drosophila S2 cells with insulin treatment made the expression of dFOXO increased, but the degradation of dFOXO was not significantly changed when dXBP1 was expressed at the same time; When S2 cells were treated with both insulin and MG132 at the same time, the degradation of dFOXO was not affected.6. dXbpl knock down resulted in a significant decrease in lipid droplet content in the fat body of drosophila, but dFoxO knock down at the same time would restore the lipid droplet content; The decreased level of triglyceride caused by dXbp1 knock down was restored by dFoxO knock down under normal feeding and starvation conditions; The anti-starvation ability decreased in dXbpl knock down group compared to the control group, while dFoxO knock down can restore this ability to some extent.7. In wings of drosophila from dFoxO over expression group, an upper longitudinal short vein disappeared in comparison with the control group; the disappeared vein reappeared in the wings of drosophila when dXBP1s was expressed at the same time.8. Drosophila larvae in dFoxO over expression group had a slow development and were not able to develop into adults, while the larvae in dXbpls and dFoxO co-over expression group were successfully develop into adults; After 15 days, no larvae survived in the dFoxO over expression group, while the number of larvae in co-over expression group was significantly increased compared to that of dFoxO over expression group.9. The lipid droplets in the fat body of the drosophila in dFoxO over expression group were significantly decreased compared to that in the control group and dXbpls over expression group, while the lipid droplets were restored in group which dFoxO and dXbp1s were co-over expressed.Conclusion:1. The activation of endoplasmic reticulum stress response is involved in the process of fat mobilization during starvation, and the endoplasmic reticulum stress protein dXBP1s in the Drosophila fat body increases the survival time of Drosophila by reducing the excessive consumption of fat under starvation condition.2. The endoplasmic reticulum stress protein dXBPl in the Drosophila fat body regulates the fat mobilization of Drosophila larvae by controlling the degradation of dFOXO protein and has the effect on the development of larvae.3. The mechanism of the negative regulation of dFOXO protein by the endoplasmic reticulum stress protein dXBP1 in Drosophila fat body is to promote the degradation of dFOXO protein under starvation condition, and the degradation process is proteasome-dependent instead of insulin-pathway dependent. More specific mechanisms remain to be further explored.