Rosiglitazone Counteracts Palmitate-induced β-cell Apoptosis By Activation Of Autophagy

The pathogenesis of type 2 diabetes has two main aspects, insulin resistance and relative impairment in insulin secretion.. Isletβ-cell apoptosis is the important reason of lacking of isletβ-cell secretion, so how to protectβ-cells has been the focus of the study. Even if we very effectivly control blood glucose,βcell function will still gradually deteriorate. A number of clinical studies has confirmed the important role ofβcell function in the development of type 2 diabetes. UKPDS Study (United Kingdom Prospective Diabetes Study), ADOPT study (A Diabetes Outcome Progression Trial) and the Belfast diet study (Belfast Diet Study) have shown that high blood sugar degradation coincided with the deterioration ofβcell function. Conventional therapy can only slow down the development of type 2 diabetes, but not prevent its development. UKPDS reported nothing to do with the treatment of type 2 diabetes,high blood sugar andβcell function is gradually deteriorated. ADOPT also reported similar results, regardless of what kind of drug therapy,βcell function is parallel with the progress of high blood sugar. The loss ofβcell function lead to the progress of high blood sugar. Studies reported that about 50%β-cell apoptosis have happed in the early stage of diabetes. Therefore, it is important to search for the mechanisms ofβcell apoptosis and find a favorable treatment to delay the progress of type 2 diabetes.Studies confirmed autophagy help cells to escape apoptosis. Autophagy is a regulated lysosomal degradation pathway. Autophagy is the cell's major regulated mechanism for degrading long-lived proteins and the only known pathway for degrading organelles. Autophagy widely exists in the normal physiological process, and maintains the intracellular homeostatisis.autophagy is the rapid adaptive response of cells to exogenous stress. Autophagy is rapidly upregulated when cells need to generate intracellular nutrients and energy (e.g., during starvation or trophic factor withdrawal), or rid themselves of damaging cytoplasmic components (e.g., during oxidative stress, infection, and accumulation of protein aggregates). Autophagy help cells to escape apoptosis, its mechanism may be autophagosome engulf damaged mitochondria, prevent the proliferation of pro-apoptotic factors such as cytochrome and apoptosis inducing factor (AIF).Recent study found that autophagy can prevent pancreaticβ-cell apoptosis, maintainβ-cell's number and internal environment. It has been reported that in STZ (Streptozotocin, STZ)-induced experimental diabetes model, the first reaction in male Wistar rat isletβcells is autophagy, which play a cell self-protection role. Similarly, Choi SE and other studies also showed that autophagy is a protective mechanism for INS-1 cells to escaping from apoptosis induced by palmitic acid. Ebato C etal found that in the standard diet, pancreatic P cells in non-diabetic mouse maintains a very low level of autophagy; but givng the high fat diet, the diabetic and non-diabetic mice P cell have significant high autophagylevel. Under the electron microscope, a large number of autophagic bodies appeared. To further study the physiological functions of autophagy, Ebato C etal, constructed pancreaticβcell autophagy-specific gene ATG7 knockout mouse model (Atg7f /f:RIP-Cre), and found that p cells showed apoptosis and compensatory proliferation was damaged during high fat diet. Increased amounts of degradated cells and accumulation of p62 protein were also found. This study shows that in response to external stress, autophagy helps cells to remove unwanted and harmful proteins to protect P cells from these harmful substances induced-apoptosis. In addition, liang j et al also confirmed that in autophagy-defect isletβcells, mitochondrial dysfunction led to lower production of ATP and NADPH2, which induced apoptosis and cell cycle arrest. These studies show that autophagy is an important protection mechanism for isletβcell.Thiazolidine dione (TZD) are a class of widely used in clinical treatment of diabetes drugs, the present study suggested it can not only lower blood sugar but also play a role in protecting pancreaticβcells, but the mechanism of protection toβcells is not clear. Literatures suggested that TZD can activate AMPK. The activation of AMPK in skeletal muscle and other organizations is considered to enhance the uptake of glucose, improve insulin resistance, but its role in pancreatic isletβcell remains controversial. In addition, the mechanism of TZD activating AMPK is not clear. some studies suggested that it was through the non-PPARy receptor pathway. Taking into account AMPK is currently thought to promote autophagy, we speculated that rosiglitazone may through the activation of AMPK to activate autophagy, which counteractsβ-cell apoptosis. The purpose of this study is:to clarify autophagy in role of the protection of rosiglitazone on pancreatic islet B cells and its mechanism. The present work consists of the following four main components: part I:the effect of rosiglitazone on palmitate-inducedβ-cellApplicating CCK-8 method, placenta-Ian detection and cloning experiments to detect the death ofβ-cell influenced by rosiglitazone and palmitate. light microscopy, AnnexinV /PI double staining, DAPI staining and TUNEL Detecting pancreaticβcells (INS-1 cells) apoptosis. The results showed that rosiglitazone + palmitate group's apoptosis is higher than the group of palmitate-induced P-cell, indicating that rosiglitazone can protect islet P-cell apoptosis induced by free fatty acid. part II:rosiglitazone activate autophagy of P-cellGFP-LC3 transfected isletβ-cells and fluorescence microscope observe the formation of punctate fluorescence; westernblot detected LC3Ⅱprotein expression, and electron microscope observe autophagosome in the isletβ-cell induced by rosiglitazone. The results showed that rosiglitazone can activateβ-cell autophagy. part III:the mechanism of rosiglitazone activating the autophagy in P-cellTesting AMPK phosphorylation of pancreaticβ-cell under the action of rosiglitazone, and detecing the autophagy change under the application of AMPK inhibitor,we determine the role of AMPK in which rosiglitazone activated autophagy in pancreaticβ-cell.The results showed that rosiglitazone increased islet P-cell AMPK phosphorylation,and after the inhibition of AMPK, autophagy was reduced. It suggested that rosiglitazone activated autophagy may be through AMPK pathway. In order to further study autophagy in the role of protection of rosiglitazone onβcells, we continued the part VI partⅥ:the effect of the inhibition of autophagy to the protective effect of rosiglitazone onβcells(1) The autophagy inhibitor (3-MA, chloroquine, or si-beclinl) pretreat pancreatic P cells, and to observe the protective effection of rosiglitazone on the P cells. Detection contents is same as the first part.(2) inhibit AMPK and observe the protective effect of rosiglitazone on P cells. Detection contents is same as the first part.The results showed that rosiglitazone did not prevent high-fat induced islet P-cell apoptosis after inhibiting autophagy or AMPK. It suggested the autophagy may one important mechanism Participating in the effection of rosiglitazone preventing the pancreatic isletβ-cell apoptosis. Thus, our results suggested that Rosiglitazone may counteract palmitate-inducedβ-cell apoptosis by activation of autophagy.