title: Tumor necrosis factor-{alpha} decreases Akt protein levels in 3T3-L1 adipocytes via the caspase-dependent ubiquitination of Akt.
abstract: TNF-alpha 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-alpha decreases Akt levels. TNF-alpha 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-alpha treatment, indicative of Akt cleavage. The broad-spectrum caspase inhibitor t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone markedly suppressed these effects of TNF-alpha. The caspase-6 inhibitor Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH(2)F 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-alpha also enhanced the association of Akt with an E3 ligase activity. Adipocytes preexposed to TNF-alpha 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-alpha. Collectively, our results suggest that TNF-alpha 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-alpha exposure leads to impaired Akt-dependent insulin signaling in adipocytes. These findings expand the mechanism by which TNF-alpha impairs insulin signaling.
authors.affiliation: Signal Transduction, Department of Internal Medicine, University of California School of Medicine, 6510 Genome and Bioscience Facility Building, 451 East Health Sciences Drive, Davis, California 95616, USA.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Medina
authors.forename: Robert R
authors.initials: RR
authors.lastname: Afsari
authors.forename: Tommer
authors.initials: T
authors.lastname: Ravid
authors.forename: S Sianna
authors.initials: SS
authors.lastname: Castillo
authors.forename: Kent L
authors.initials: KL
authors.lastname: Erickson
authors.forename: Tzipora
authors.initials: T
authors.lastname: Goldkorn
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D007328:Insulin / Q000378:metabolism; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D007328:Insulin; D009842:Oligopeptides; D015513:Oncogene Proteins; D061988:Proteasome Inhibitors; D011518:Proto-Oncogene Proteins; D014409:Tumor Necrosis Factor-alpha; D025801:Ubiquitin; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; C491753:Akt3 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020169:Caspases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease