Abstract
Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (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 is 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 stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by β-methylcyclodextrin that abolishes 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. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs.
Highlights
Glucose and other reducing sugars can react non-enzymatically with the amino groups of proteins and lipids to form Schiff bases
Since receptor for advanced glycation end products (RAGE) and several signaling proteins are localized in caveolae and caveolins regulate a variety of signaling pathways, we examined the involvement of Cav-1 in RAGE-mediated Akt activation in 3T3-L1 cells
Our results showed that NAD(P)H oxidase, Src and IGF-1 receptor transactivation are involved in the activation of Akt by advanced glycation end products (AGEs). 3T3-L1 cells can be induced to differentiate into adipocytes by IBMX, dexamethasone, and insulin/IGF-1
Summary
Glucose and other reducing sugars can react non-enzymatically with the amino groups of proteins and lipids to form Schiff bases. The Schiff bases are slowly rearranged to form Amadori products which undergo further rearrangements, oxidation, dehydration and condensation resulting in compounds called advanced glycation end products (AGEs). AGEs are formed in normal physiological condition. The formation and accumulation of AGEs are accelerated in tissues from aged individuals and diabetic patients [1,2,3,4,5,6]. AGEs exert their cellular functions via the interaction with their specific receptor, the receptor for advanced glycation end products (RAGE) [7]. Binding of AGEs to RAGE activates a variety of signaling proteins and downstream transcription factors including Src, NAD(P)H oxidase, Ras/ERK1/2, PI3K/PDK1/Akt, p38
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