Regulation Of Adiponectin On Apoptosis Of 3T3-L1 Adipocyte And Its Molecular Mechanism

Adiponectin, the adipocyte-specific secreted cytokine, is abundant in blood. The regulatory mechanisms of adiponectn on adipose differentiation, muscle, pancreatic islet cells differentiation and metabolic and mitochondrial function have been deeply studied. Apoptosis refers to the programmed cell death process that activated and regulated by a variety of genes to maintained homeostasis. Recently, the regulation of adipose apoptosis is considered to be one of the most important factors that affect the deposition of body fat. However, few researches are concentrating on adipose apoptosis. Studies have shown that adiponectin can regulate apoptosis of muscle cells and pancreatic β cells, but how it affects adipose apoptosis remains unclear.To determine the effect and mechanism of adiponectin on adipose apoptosis, we forced expression of adiponectin and interference of adiponectin on 3T3-L1 adipocytes in this experiment to detect its regulation role on adipose apoptosis. In addition, palmitic acid, serum starvation and tunicamycin were used to treat cells to establish the apoptosis models to further verify the effects of adiponectin on adipose apoptosis. Furthermore, in order to dissect the mechanism about adiponectin on adipose apoptosis, we detected the levels change of signaling pathways. The followings are the results we obtained:1. Adiponectin suppressed the apoptosis of 3T3-L1 adipocytes. After forced expression of adiponectin or stable knock down of adiponectin, Adipocytes were treated with palmitic acid to establish the apoptosis model. Realtime-PCR results showed the level of Caspase3, the marker gene of apoptosis, and other factors functioned in the process of apoptosis were elevated along with the treating time(P <0.05). Hochest33258 staining showed the morphological changes of adipocytes apoptosis, and adiponectin significantly reduced the ratio of apoptotic cells. By Annexin V-FITC / PI staining and flow cytometry, we further observed adiponectin could inhibit adipocytes early phase and late phase apoptosis.2. Adiponectin regulated the mitochondrial pathway of apoptosis. The data of JC-1 staining of mitochondrial membrane potential showed over expression of adiponectin increased mitochondrial membrane potential significantly, while interference of adiponectin had the opposite result(p < 0.01). Immunofluorescence staining of Cyt C indicated adiponectin overexpression significantly inhibited the release of Cyt C. Additionally, Western Blot data showed the apoptosis related genes were lower in overexpression of adiponectin group and adiponectin inhibited pro-apoptotic factors Bax, Apaf-1 and the expression of cleaved Caspase9, cleaved Caspase3, while increased the levels of anti-apoptotic factor Bcl-2 expression(P <0.01). These results collectively showed that adiponectin can inhibit Cyt C/ Apaf-1 / Caspase9 / Caspase3 pathway which is a classic mitochondrial apoptotic pathway. Moreover, we detect ERK1/2 signaling pathway and we found overexpression of adiponectin significantly increased ERK1/2 phosphorylation levels, indicating that adiponectin inhibited adipocyte apoptosis by activating ERK signaling pathway.3. Adiponectin regulated adipocyte cycle arrest and apoptosis. By serum starvation-induced apoptosis, Realtime-PCR detection showed the expression of key factors of cell cycle and apoptosis were significantly increased along with the treatment time(P <0.05). Flow cytometry analysis of cell cycle indicated forced expression of adiponectin significantly decreased the ratio of cells under G0/G1 phase compared with that in the control group. And the cells under S phase were remarkably higher with adiponectin overexpression. We then come to the conclusion that adiponectin promoted the cells transition from G0/G1 to S phase. Western Blot results showed that overexpression of adiponectin significantly decreased the protein levels of p27 and p53, while the expression of Cylin E was significantly higher. And the expression of Caspase3 and cleaved PARP were lower than that in the control group. These results suggested that adiponectin improved cell cycle arrest. However, further studies are needed to explore the underlying mechanism.4. Adiponectin regulated ER stress and apoptosis. Tunicamycin was used to induce endoplasmic reticulum stress in our experiment, the marker genes of endoplasmic reticulum stress and apoptosis were detemined. Western Blot showed that forced expression of adiponectin significantly blocked the expression of CHOP, GRP78, PERK, and reduces the expression of apoptotic factor PARP(P <0.05). JNK signaling pathway detection indicated adiponectin inhibited JNK phosphorylation, suggesting adiponectin alleviated endoplasmic reticulum stress via the inhibition of CHOP / JNK pathway and thus attenuated cell apoptosis induced by endoplasmic reticulum stress.In summary, this study demonstrated adiponectin inhibited adipocyte apoptosis by establishing three apoptosis models. Then explored the effects of adiponectin on three different kinds of apoptosis: mitochondrial pathway related apoptosis, cell cycle arrest and endoplasmic reticulum stress-induced cell apoptosis. And we found ERK and JNK signaling pathways were involved in the regulation of adiponectin on cell apoptosis.