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Tumor Necrosis Factor- Decreases Akt Protein Levels in 3T3-L1 Adipocytes via the Caspase-Dependent Ubiquitination of Akt

Signal Transduction, Department of Internal Medicine (E.A.M., R.R.A., T.R., S.S.C., T.G.), and Department of Cell Biology and Human Anatomy (E.A.M., K.L.E.), University of California School of Medicine, Davis, California 95616

Address all correspondence and requests for reprints to: Dr. Tzipora Goldkorn, Signal Transduction, 6510 Genome and Bioscience Facility Building, 451 East Health Sciences Drive, Davis, California 95616. E-mail: ttgoldkorn{at}ucdavis.

TNF- is a mediator of insulin resistance in sepsis, obesity, and type 2 diabetes and is known to impair insulin signaling in adipocytes. Akt (protein kinase B) is a crucial signaling mediator for insulin. In the present study we examined the posttranslational mechanisms by which short-term (<6-h) exposure of 3T3-L1 adipocytes to TNF- decreases Akt levels. TNF- treatment both increased the ubiquitination of Akt and decreased its protein level. The decrease in protein was associated with the presence of an (immunoreactive) Akt fragment after TNF- treatment, indicative of Akt cleavage. The broad-spectrum caspase inhibitor t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone markedly suppressed these effects of TNF-. The caspase-6 inhibitor Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH₂F potently suppressed Akt ubiquitination, degradation, and fragment formation, whereas the proteasome inhibitor Z-Leu-Leu-Leu-CHO modestly attenuated the decline in Akt levels. Exposure to TNF- also enhanced the association of Akt with an E3 ligase activity. Adipocytes preexposed to TNF- for 5 h and then stimulated with insulin for 30 min exhibited decreased levels of Akt, phosphorylated Akt, as well as phosphorylated Mdm2, which is a known direct substrate of Akt, and glucose uptake. Caspase inhibition attenuated these inhibitory effects of TNF-. Collectively, our results suggest that TNF- induces the caspase-dependent degradation of Akt via the cleavage and ubiquitination of Akt, which results in its degradation through the 26S proteasome. Furthermore, the caspase- and proteasome-mediated degradation of Akt due to TNF- exposure leads to impaired Akt-dependent insulin signaling in adipocytes. These findings expand the mechanism by which TNF- impairs insulin signaling.

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