Mechanisms of the Activation of PI3-Kinase/Akt and Promotion of Adipogenesis by Advanced Glycation Endproducts (AGEs)

• Main Authors: Shu-Jung Yang, 楊淑茸
• Other Authors: Shi-Chuan Chang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/36852429804385822117

博士 === 國立陽明大學 === 藥理學研究所 === 101 === Aging and diabetes mellitus (DM) are characterized by hyperglycemia. High glucose interacts with proteins and other substances and forms advanced glycation end products (AGEs). AGEs are therefore accumulated in tissues from the elderly and diabetic patients. However, the precise role of AGEs in aging and diabetic complications remains to be explored. AGEs activate signaling proteins such as Src, NAD(P)H oxidase, PI3/PDK1/Akt, Ras/ERK1/2, and p38 MAPK. However, the mechanisms by which AGEs activate these signaling proteins are still unclear. Therefore, my study aimed to clarify the mechanism of intracellular signal transduction and of adipogenesis elicited by AGEs in 3T3-L1 preadipocytes with a specific focus on the PI3-kinase-PDK1-Akt activation. I found that AGEs activated Akt in a dose- and time-dependent manner in 3T3-L1 cells. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or NAD(P)H oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src was downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorβsubunit (IGF-1Rβ) on Tyr1135/1136 which was sensitive to PP2, indicating that AGEs stimulated Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolished the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rβ and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. I further found that AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes by elevating PPARγ and C/EBPα, the master transcription factors governing adipogenesis. AGEs treatment also increased levels of markers for differentiated adipocytes, aP2 and GPDH activity. In contrast, addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis by reducing PPARγ and aP2 levels. These results suggest that AGEs promote adipogenesis by trans-activating IGF-1 receptor, PI3-kinase and Akt. For comparison, I also investigated the signaling mechanism on the activation of ERK1/2 and PI3-kinase-Akt by plumbagin (a strong oxidizing radical inducer) derived from the roots of Plumbagon zeylanica. Plumbagin has been shown to chronically activate ERK1/2 and inhibit Akt activity in cancer cells. However, the acute effects of plumbagin on ERK1/2 and Akt activities remain unknown. Exposure of 3T3-L1 cells to plumbagin generated superoxide and activated both ERK1/2 and Akt. The plumbagin-stimulated ERK1/2 and Akt activities were sensitive to NAC, Tiron, DPI and superoxide dismutase mimetic MnTBAP. Plumbagin-stimulated ERK1/2 activity was attenuated by the MEK1/2 inhibitor PD98059 and Ras inhibitor manumycin A, whereas plumbagin-stimulated Akt activity was blocked by the PI3 inhibitor LY294002. Both plumbagin-stimulated ERK1/2 and Akt activities were attenuated by PP2, a Src inhibitor. Interestingly, inhibition of phosphatidylinositol 3-kinase (PI3-kinase), but not Akt, activity leaded to attenuation of plumbagin-stimulated ERK1/2 activity. These results suggest that plumbagin activates NAD(P)H oxidase, Src, and PI3, and that the activated PI3 or PDK1 subsequently stimulates Akt and Ras-Raf-MEK1/2-ERK1/2 in 3T3-L1 cells.