Tumor Necrosis Factor- Decreases Akt Protein Levels in 3T3-L1 Adipocytes via the Caspase-Dependent Ubiquitination of Akt
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内分泌学杂志 2005年第6期
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@ucdavis.
Abstract
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.
Introduction
THE PROINFLAMMATORY CYTOKINE TNF- is well known for its role in contributing to insulin resistance during infections, surgery, and trauma. The impaired ability of muscle and adipose tissue to respond to insulin frequently leads to hyperglycemia and hyperlipidemia in those inflammatory states. TNF- is also implicated as a mediator of insulin resistance in obesity and type 2 diabetes. Adipose tissue production of that cytokine is enhanced in insulin-resistant obese or diabetic animals and humans (1, 2), and administration of TNF- receptor IgG fusion protein or soluble TNF--binding protein, which neutralize TNF-, improved insulin responsiveness in obese and insulin-resistant animals (1, 3).
Several studies have demonstrated that TNF- impairs insulin responsiveness in cultured adipocytes. Characterization of the defect caused by TNF- has been elusive, because the cytokine interferes with insulin signaling at multiple levels. Although one study reported that acute exposure (15–120 min) of 3T3-L1 adipocytes to TNF- enhanced insulin-stimulated IRS-1 tyrosine phosphorylation and binding to phosphoinositide 3-kinase (PI3 kinase) (4), several demonstrated that acute treatment (<6 h) of human or 3T3-L1 adipocytes with TNF- decreased insulin-stimulated insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, PI3 kinase activity, and glucose transport (5, 6, 7, 8). The demonstrations that serine/threonine phosphorylation of IRS-1 impaired insulin signaling (9) and that TNF- induced serine/threonine phosphorylation of IRS-1 (10) suggest a mechanism for how the cytokine might attenuate IRS-1 function. Prolonged exposure to TNF- was shown to inhibit both IR autophosphorylation and IRS-1 phosphorylation (11). Chronic treatments also decreased transcript and protein levels of molecules that mediate the effects of insulin, including IRS-1, cyclic-nucleotide phosphodiesterase-3B (PDE3B), and glucose transporter-4 (12, 13); reduced amounts of these proteins resulted in impaired insulin responses. Short-term (1- to 6-h) treatment with TNF- also reduced Akt (also known as protein kinase B) protein levels in 3T3-L1 adipocytes (14), although the mechanism for its depletion or its effect on insulin action has not been explored.
Insulin triggers the autophosphorylation of intracellular tyrosine residues on the insulin receptor, which results in recruitment of IRS and activation of the lipid kinase PI3 kinase. PI3 kinase then generates 3'-phosphatidylinositol lipids that recruit Akt to the plasma membrane, where it is phosphorylated and thereby activated by 3'-phosphoinositide-dependent protein kinase-1 and -2. The Akt family consists of three highly homologous isoforms (Akt1, Akt2, and Akt3), and it mediates a number of insulin-induced functions, including glucose uptake, suppression of lipolysis via activation of PDE3B, and inhibition of glycogen synthase kinase-3, which enables activation of glycogen synthase (15). These responses are impaired in animals or humans with sepsis, obesity, and type 2 diabetes (16, 17, 18, 19, 20, 21), and TNF- has been shown to impair insulin-mediated glucose uptake in cultured adipocytes (5, 11, 12, 22). Because TNF- can decrease Akt expression in adipocytes, diminished Akt protein levels probably contribute to the impairment of insulin responses mediated by the PI3 kinase/Akt pathway in conditions noted for systemic inflammation.
In the present study we examined the mechanism by which TNF- decreases Akt protein levels in 3T3-L1 adipocytes. We report that TNF- decreases Akt expression via the caspase-dependent cleavage of Akt1 and the caspase-dependent ubiquitination of Akt1 and Akt2, which leads to their degradation by the 26S proteasome. We also demonstrate that inhibition of caspases or the 26S proteasome attenuates the effect of TNF- to impair Akt-dependent insulin signaling.
Materials and Methods
Reagents
TNF- was purchased from R&D Systems, Inc. (Minneapolis, MN). Cycloheximide (CHX), actinomycin (ACT), Ac-Leu-Leu-arginal (leupeptin), chloroquine, and N-acetyl-Leu-Leu-norleucinal were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Z-Leu-Leu-Leu-CHO (MG132), t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone (Boc-D-FMK), Z-Tyr-Val-Ala-Asp(OMe)-CH2F (Z-YVAD-FMK), Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH2F (Z-VDVAD-FMK), Z-DEVD-FMK, Z-Trp-Glu(OMe)-His-Asp(OMe)-CH2F (Z-WEHD-FMK), Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH2F (Z-VEID-FMK), Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (Z-IETD-FMK) and Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (Z-LEHD-FMK) were acquired from Calbiochem (San Diego, CA) and dissolved in Me2SO (Sigma-Aldrich Corp.) before their addition to the cell cultures. The antibodies used were polyclonal anti-Akt, polyclonal anti-phospho-Akt (Ser473), polyclonal anti-phospho-Mdm2 (Ser166), and anti-biotin, horseradish peroxidase-linked (Cell Signaling, Beverly, MA); polyclonal anti-Akt1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); polyclonal anti-Akt1, polyclonal anti-Akt2, polyclonal anti-Akt3, and polyclonal anti-p85 (PI3 kinase; Upstate Biotechnology, Lake Placid, NY); and monoclonal anti-ubiquitin (anti-Ub; Covance Research Products, Inc., Berkeley, CA).
3T3-L1 cell culture
3T3-L1 fibroblasts were grown at 37 C in 5% CO2 until 2 d post confluence in high glucose DMEM (Invitrogen Life Technologies, Inc., Grand Island, NY) supplemented with 10% calf serum (HyClone, Logan, UT), and then differentiation was induced by a 48-h incubation with DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.), dexamethasone (0.25 mM; Sigma-Aldrich Corp.), methylisobutylxanthine (100 μM; Sigma-Aldrich Corp.), and insulin (10 μg/ml; Sigma-Aldrich Corp.). Cells continued to differentiate for another 5–9 d in DMEM supplemented with insulin (10 μg/ml) until more than 90% of the cells demonstrated the adipocyte morphology, as assessed by Oil Red O staining. Before treatment, cells were incubated in low glucose DMEM supplemented with 10% calf serum for 48 h, followed by incubation in low glucose DMEM supplemented with 0.5% calf serum for 24 h.
RNA extraction, RT, and real-time PCR
Total RNA was isolated from adipocytes with TRIzol (Invitrogen Life Technologies, Inc.) as previously described (23). Extracted RNA for each sample was quantified with RiboGreen (Molecular Probes, Eugene, OR), and 2 μg were reverse transcribed with SuperScript II Plus RNase H– Reverse Transcriptase using an oligo(deoxythymidine) primer (Invitrogen Life Technologies, Inc.). The LightCycler (Roche, Indianapolis, IN) was used for real-time PCR amplification. For real-time PCR, the following oligonucleotide primers were synthesized (Invitrogen Life Technologies, Inc.) using the cDNA sequences (GenBank sequence database of the National Center for Biotechnology Information) for murine Akt1 (M94335), Akt2 (U22445), and Akt3 (AF124142.1): 5'-GGCGTGGTCATGTACGAGATG-3' (Akt1 sense), 5'-TGAGCTGTGAACTCCTCATCGA-3' (Akt1 antisense), 5'-CATTCTTATGGAGGAGATCCGCTT-3' (Akt2 sense), 5'-GGTCCAGGCTGTCATATCGGT-3' (Akt2 antisense), 5'-AAGTATGACGACGACGGCATG-3' (Akt3 sense), and 5'-AGCAACAGCATGAGACCTTAGACTG-3' (Akt3 antisense). The sequences for the ?-actin oligonucleotide primers were previously reported (24). Real-time PCR efficiencies were calculated for each target gene (E = 10–1/slope), and all were determined to be approximately equal. Each reaction mixture consisted of 2.0 μl template DNA, 4 mM MgCl2, 0.5 μM of each primer, and LightCycler-DNA Master SYBR Green I in a total volume of 20 μl (Roche). The template DNA was diluted 50-fold before amplification with a protocol that consisted of 40 cycles with denaturation at 95 C for 1 sec, annealing at 56 C for 5 sec, and sequence extension at 72 C for 15 sec. A crossing point (Cp) for each sample was generated using the fit point method in the LightCycler software (Roche). Relative gene expression (over untreated cells) was obtained after normalization to ?-actin and determination of the difference between Cp in treated and untreated cells using the 2–Cp formula (25). Product identity was confirmed initially by sequence analysis and subsequently with the detection of a single product-specific melting temperature for each gene of interest with the LightCycler melting curve analysis program.
Immunoprecipitation and Western blotting
Lysates were extracted in solubilization buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, 0.1% SDS, 5 mM N-ethylmalemide (Sigma-Aldrich Corp.), and protease and phosphatase inhibitor cocktail (Sigma-Aldrich Corp.). Lysates were cleared by centrifugation (16,000 x g) for 45 min, and protein concentrations were measured using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of protein were resolved by 10% SDS-PAGE. For immunoprecipitations, 500 μg protein were incubated overnight at 4 C with the appropriate antibody, then precipitated the next day for 2 h with protein G (Upstate Biotechnology, Inc.). Immunoprecipitates were resolved by 5–15% SDS-PAGE. Resolved proteins were transferred onto a nitrocellulose membrane and then blocked for 1 h in Tris-buffered saline, pH 7.5, containing 0.5% Tween 20 and 5% milk. Membranes were incubated overnight at 4 C with the appropriate primary antibody. The next day, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h, and the immunoreactive bands were detected with an enhanced chemiluminescent reagent (Pierce Chemical Co., Rockford, IL). Images of the bands were photographed, and densitometry was performed with National Institutes of Health Image software.
In vitro ubiquitination assay
Immunoprecipitates were generated as described above, except that neither 0.1% SDS nor N-ethylmalemide was present in the lysis buffer. Beads loaded with Akt were washed with PBS, then incubated with 20 μl reaction mixture [300 mM HEPES (pH 7.2–7.6), 20 mM ATP, 50 mM MgCl2, and 2 mM dithiothreitol] and 1 μg biotin-N-terminal Ub (Boston Biochem, Inc., Cambridge, MA) that included either 40 ng E1 (Affinity Research, Ltd., Exeter, UK) and 600 ng each of UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7, and UbcH10 (Affinity Research, Ltd.) or PBS. Samples were incubated and rocked for 1 h at 30 C. Reactions were stopped and incubated for 20 min with sodium dodecyl sulfate sample buffer containing 360 mM iodoacetamide, then analyzed by immunoblotting as described above.
Glucose uptake by 3T3-L1 adipocytes
Uptake of 2-deoxy-D-[3H]glucose (Amersham Biosciences, Arlington Heights, IL) was assessed in 3T3-L1 adipocytes differentiated in six-well plates as previously described (26) with minor modifications. After the treatments indicated in the figure legends, adipocytes were washed twice with 2 ml KRH buffer (129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.8 mM CaCl2, and 20 mM HEPES, pH 7.4), then incubated in 1 ml/well KRH buffer containing 200 nM insulin for 30 min at 37 C. Afterward, the cells were incubated in 1 ml KRH buffer containing 0.1 mM 2-deoxyglucose and 1 μCi/ml 2-deoxy-D-[3H]glucose for 5 min. Adipocytes were then washed three times with ice-cold PBS and solubilized in 1.0 ml 0.5 N NaOH and 0.1% sodium dodecyl sulfate. [3H]Glucose uptake was detected in 3 ml scintillant containing 0.3 ml lysate using a Beckman LS6500 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Nonspecific deoxyglucose uptake (<10% of the total) was measured in the presence of 20 μM cytochalasin B and was subtracted from each determination to yield insulin-specific glucose uptake. Aliquots of the lysates were also subjected to protein concentration determination (Bio-Rad Laboratories).
Results
TNF- reduces Akt protein levels
3T3-L1 adipocytes were treated with TNF-, and the effects on Akt protein levels were characterized. Cell lysates were clarified, resolved by SDS-PAGE, and then immunoblotted with polyclonal anti-Akt. Incubation with TNF- for 5 h dose-dependently decreased Akt levels (Fig. 1A). TNF- also inhibited Akt protein levels in a time-dependent manner (Fig. 1B). TNF- did not affect the protein levels of the PI 3-kinase component p85. This is consistent with a previous report that TNF- treatment with a similar concentration and exposure period did not affect p85 protein levels in 3T3-L1 adipocytes (27). We therefore used p85 as a loading control for our subsequent studies. Immunoblotting with the polyclonal anti-Akt also revealed a low molecular mass immunoreactive band that migrated at approximately 44 kDa, which is in the predicted molecular mass range for a caspase-cleaved Akt fragment (40–50 kDa) (28).
FIG. 1. TNF- decreases Akt protein levels. A, Upper panel, Fully differentiated 3T3-L1 adipocytes were incubated with 0.06–6.0 nM TNF- for 5 h. Cell lysates were prepared in lysis buffer containing 1% Nonidet P-40. Lysates were clarified, resolved (25 μg/lane) by 10% SDS-PAGE gel, and then immunoblotted with a total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. Lower panel, The graph represents densitometry of Akt levels as a percentage of the control values in the blot shown (upper panel). A representative blot of at least three independent experiments is shown. B, Left panel, 3T3-L1 adipocytes were treated with 3 nM TNF- for 2–6 h. Cell lysates were resolved by 10% SDS-PAGE gel and immunoblotted with total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. Right panel, The graph represents densitometry of Akt levels as a percentage of the control values in the blot shown (left panel). A representative blot of at least three independent experiments is shown.
Reductions in Akt protein levels by TNF- involve caspases and the 26S proteasome
A recent study demonstrated by microarray analysis that exposure of 3T3-L1 adipocytes to TNF- for 24 h suppressed Akt2 (–3.6-fold), but not Akt1, gene expression (14). Using real-time RT-PCR, we observed that exposure to TNF- for 6 h suppressed mRNA levels of both Akt1 and Akt2 in 3T3-L1 adipocytes (Fig. 2A). To determine whether posttranslational effects were also involved in decreasing Akt levels during exposures to TNF-, adipocytes were pretreated with either the transcription inhibitor ACT or the translation inhibitor CHX before incubation with TNF-. Both ACT and CHX enhanced the TNF--induced decrease in Akt (Fig. 2B). Thus, the TNF--induced suppression of Akt protein levels in 3T3-L1 adipocytes involves both transcriptional and posttranslational mechanisms. To investigate the posttranslational effects of TNF- on Akt expression, subsequent experiments used CHX to control for transcriptional changes.
FIG. 2. TNF- decreases Akt1 and Akt2 mRNA levels and decreases Akt protein levels in the presence of a transcriptional or translational inhibitor with involvement of caspase(s) and the 26S proteasome. A, 3T3-L1 adipocytes were exposed to 3 nM TNF- for 3–6 h. Akt1, Akt2, and Akt3 mRNA levels were determined by real-time RT-PCR as described in Materials and Methods. B, 3T3-L1 adipocytes were preincubated with 10 μg/ml ACT or 0.5 mM CHX for 1 h, then exposed to 3 nM TNF- for 5 h. ACT and CHX were used as inhibitors of transcription and protein translation, respectively. Cell lysates were resolved by 10% SDS-PAGE gel and immunoblotted with a total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. A representative blot of at least three independent experiments is shown. C, Left panel, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h or with 10 μM MG132, 250 μM leupeptin, 50 μM BOC-D-FMK, or 150 μM chloroquine for 1 h, then exposed to 3 nM TNF- for 5 h. Cell lysates were prepared as described in Fig 1. Twenty-five micrograms of lysate were then resolved by 10% SDS-PAGE gel and immunoblotted with a total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. Right panel, The graph represents densitometry of Akt levels in the blot shown in the left panel. A representative blot of at least three independent experiments is shown.
Posttranslational strategies employed by cells to decrease the levels of a given protein include proteasome-, calpain-, caspase-, and lysosome-mediated degradation. To test whether these pathways mediated the effect of TNF- to decrease Akt expression, cells were pretreated with specific inhibitors of these pathways (Fig. 2C). Pretreatment with the broad-spectrum caspase inhibitor Boc-D-FMK completely blocked the ability of TNF- to decrease Akt protein levels. A modest protection from a decrease in Akt levels was also observed with pretreatment with the 26S proteasome inhibitor, MG132 (29). However, the thiol protease inhibitor leupeptin (30) and the lysosomal inhibitor chloroquine (31) were unable to stabilize Akt levels. Boc-D-FMK also completely suppressed the TNF--induced generation of the approximately 44-kDa band. These data suggest that the observed low molecular bands generated in response to TNF- probably resulted from caspase-mediated Akt cleavage. Therefore, caspases and, to a lesser extent, the 26S proteasome are involved in the posttranslational down-regulation of Akt by TNF- in 3T3-L1 adipocytes.
Ubiquitinated Akt is increased by TNF- and suppressed by caspase inhibition
Proteins that have attached to them several molecules of the 76-amino acid polypeptide called Ub are efficiently targeted for degradation by the 26S proteasome (32). Because our data suggested proteasome involvement in Akt degradation due to TNF- exposure, we next determined whether TNF- increased the association of Ub with Akt. Akt was immunoprecipitated with polyclonal anti-Akt from lysates of adipocytes that were pretreated with CHX and then exposed to TNF- for up to 5 h. Immunoprecipitated protein was resolved by SDS-PAGE, then immunoblotted with a monoclonal antibody against Ub; the association of Ub with Akt results in the appearance of a high molecular weight protein ladder/smear. Ubiquitination of Akt was clearly increased after 5-h incubation with TNF- (Fig. 3A), and the levels of immunoprecipitated Akt were concomitantly decreased.
FIG. 3. TNF- induces caspase-dependent Akt ubiquitination. A, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h, then exposed to 3 nM TNF- for 0.5–5 h. Cell lysates were prepared as usual, except that 5 mM N-ethylmaleimide and 0.1% SDS were added to the lysis buffer. Cell lysates were immunoprecipitated with total anti-Akt antibody. Immunoprecipitates were resolved by 5–15% SDS-PAGE and immunoblotted with anti-Ub antibody (upper panel). The membrane was then stripped and immunoblotted with a total anti-Akt antibody (lower panel). B and C, Differentiated 3T3-L1 adipocytes were preincubated with 0.5 mM cycloheximide for 1 h, preincubated with 50 μM BOC-D-FMK or 10 μM MG132 for 1 h, then exposed to 3 nM TNF- for 5 h. Cell lysates were prepared as usual, except that 5 mM N-ethylmaleimide and 0.1% SDS were added to the lysis buffer. Cell lysates were immunoprecipitated with anti-Akt1 (B) or anti-Akt2 (C) antibody. Immunoprecipitates were resolved by 5–15% SDS-PAGE gel, then immunoblotted with an anti-Ub antibody (upper panel). The membrane was subsequently stripped and reprobed with an anti-Akt1 or anti-Akt2 antibody (lower panel). Representative blots of at least three independent experiments are shown for A, B, and C.
Recent studies indicate that the three isoforms of Akt can be differentially regulated by particular stimuli (33, 34, 35, 36). Adipocytes have been demonstrated to express more Akt2 than Akt1 and extremely little Akt3 (36, 37, 38, 39). Akt2 is required for insulin responsiveness in adipocytes, and Akt1 provides significant functional redundancy; the role of Akt3 appears insignificant (40). Our results heretofore do not indicate whether the two isoforms that are critical to insulin responsiveness are both decreased by TNF- or whether the caspase and Ub/proteasome pathways are involved in the degradation of both Akt1 and Akt2. Therefore, we sought to characterize how the two isoforms were affected by TNF-. Lysates from adipocytes pretreated with CHX and then exposed to TNF- for 5 h were subjected to immunoprecipitation with isoform-specific antibodies and subsequent Western blot analysis with anti-Ub or anti-Akt. TNF- increased the ubiquitination of Akt1 and Akt2 (Fig. 3, B and C, upper panels) and decreased levels of both isoforms (Fig. 3, B and C, lower panels). Although the low molecular weight fragment was detected with only Akt1 immunoprecipitation, we are presently unable to rule out the possibility that caspases also cleaved Akt2. Broad caspase inhibition markedly decreased the accumulation of ubiquitinated Akt1 and Akt2 due to TNF- exposure, stabilized the two isoforms, and prevented accumulation of the Akt1 fragment. Proteasome inhibition attenuated the decline in Akt1 levels due to TNF- exposure and protected Akt2. Thus, TNF- regulates Akt1 and Akt2 in a similar manner by inducing the caspase-dependent ubiquitination and 26S proteasome-mediated degradation of both isoforms.
Caspase-6 is a key mediator for TNF--induced ubiquitination and cleavage of Akt1
Because broad caspase inhibition prevented the TNF--induced degradation and ubiquitination of Akt1 and Akt2, we tested the involvement of particular initiator, effector, and inflammatory caspases. Our data clearly showed that Akt1 was cleaved by caspases (Fig. 3B); thus, the following experiments were designed to detect Akt1 cleavage and ubiquitination. Adipocytes were pretreated with CHX and a panel of caspase inhibitors, then exposed to TNF-. Lysates were subjected to immunoprecipitation with polyclonal anti-Akt1 and subsequent Western blot analysis. As previously demonstrated, the broad-spectrum caspase inhibitor Boc-D-FMK completely inhibited Akt1 ubiquitination and the decline in Akt1 protein levels (Fig. 4A, upper and lower panels, respectively, lane B). The caspase-6 inhibitor, Z-VEID-FMK, inhibited both TNF--induced Akt1 ubiquitination (Fig. 4A, upper panel, and Fig. 4B, lane 6) and degradation (Fig 4A, lower panel, lane 6) to nearly the same extent as the broad-spectrum caspase inhibitor. None of the other caspase inhibitors was able to suppress both TNF--induced Akt1 ubiquitination and degradation, although caspase-8 inhibition appears to have reduced Akt1 ubiquitination. These results indicate that caspase-6 is probably a potent mediator of TNF- to increase Akt1 ubiquitination and cleave Akt1.
FIG. 4. TNF- induced Akt ubiquitination and degradation are attenuated by caspase-6 inhibition. A, Before TNF- exposure for 5 h, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h and preincubated for 1 h with 50 μM of the broad-spectrum caspase inhibitor BOC-D-FMK (lane B), 50 μM of the caspase-1 inhibitor Z-YVAD-FMK (lane 1), 50 μM of the caspase-2 inhibitor Z-VDVAD-FMK (lane 2), 50 μM of the caspase-3 inhibitor Z-DEVD-FMK (lane 3), 50 μM of the caspase-5 inhibitor Z-WEHD-FMK (lane 5), 50 μM of the caspase-6 inhibitor Z-VEID-FMK (lane 6), 50 μM of the caspase-8 inhibitor Z-IETD-FMK (lane 8), or 50 μM of the caspase-9 inhibitor Z-LEHD-FMK (lane 9). Cell lysates were prepared as usual, except that 5 mM N-ethylmaleimide and 0.5% SDS were added to the lysis buffer. Cell lysates were immunoprecipitated with anti-Akt1 antibody. Immunoprecipitates were resolved by 5–15% SDS-PAGE gel, then immunoblotted with an anti-Ub antibody. The membrane was subsequently stripped and reprobed with an anti-Akt1 antibody. B, Densitometric analysis for levels of ubiquitinated Akt1 (Ub-Akt1). A representative blot of at least two independent experiments is shown.
We verified the physiological relevance of the observed posttranslational effects of TNF- by examining the roles of caspases and the 26S proteasome in reducing Akt expression in the absence of CHX. The results were similar, except that proteasome inhibition caused protected Akt to accumulate in the insoluble cellular fraction (data not shown). This phenomenon resembles the reported effect of proteasome inhibition to cause ubiquitinated Raf-1, receptor-interacting protein, and Akt accumulation in detergent-insoluble fractions (41, 42, 43).
TNF- increases the E3 ligase activity associated with Akt
We next examined the role of TNF- in the induction of the Ub machinery. Ub is first activated via ATP-dependent formation of a thiol ester bond with a cysteine on a Ub-activating enzyme (E1), transferred to a cysteine on a Ub-conjugating enzyme (E2), and then it is covalently attached to a lysine on the protein to be degraded in a reaction catalyzed by a Ub-protein ligase (E3) (32). We hypothesized that TNF- induces the ubiquitination of Akt by enhancing the association of Akt with an E3 ligase. To test this possibility, lysates from cells incubated with TNF- for 1.5 or 3.0 h were immunoprecipitated with polyclonal anti-Akt, and the resulting immunocomplexes were incubated with biotinylated Ub and ATP and with or without E1 and E2 for 1 h. The reaction mixtures were resolved by SDS-PAGE and then immunoblotted with anti-biotin antibody or monoclonal anti-Akt1. Reaction mixtures derived from 3T3-L1 adipocytes that were incubated with TNF- for 1.5 h, but not 3.0 h, demonstrated an increased ability, compared with those from untreated cells, to generate Akt that was associated with biotinylated Ub (Fig. 5). The result at 3.0 h is probably due to the decreased amount of Akt that was immunoprecipitated from the lysate of adipocytes exposed to TNF-. Therefore, because the levels of immunoprecipitated Akt were the same for both TNF--treated (1.5 h) and untreated cells, these data indicate that TNF- enhanced the association of an E3 ligase with Akt. As expected, these data also demonstrate the requirement for E2, E1, and ATP for the in vitro ubiquitination reaction.
FIG. 5. TNF- enhances the E3 ligase activity associated with Akt. 3T3-L1 adipocytes were incubated with 3 nM TNF- for 1.5 or 3 h. Cell lysates were prepared as usual. Cell lysates (500 μg) were immunoprecipitated with total anti-Akt antibody. Beads loaded with Akt were incubated with rocking for 1 h at 30 C in a reaction mixture that included ATP, E1, and E2 or ATP and PBS (see Materials and Methods for details). Immunoprecipitates were resolved by 5–15% SDS-PAGE and immunoblotted with -biotin antibody. The membrane was subsequently stripped and reprobed with an anti-Akt1 antibody. Representative blots of at least three independent experiments are shown.
Caspase inhibition improves insulin signaling via Akt in adipocytes exposed to TNF-
TNF- is implicated as a mediator of sepsis- and obesity-related insulin resistance. Therefore, we tested whether diminished Akt levels due to TNF- exposure would impair Akt-dependent signaling. When adipocytes were pretreated with CHX, exposed to TNF- for another 5 h, and then stimulated with insulin for 30 min, the levels of Akt and phosphorylated Akt were markedly suppressed (Fig. 6A). Both Boc-D-FMK and MG132 impaired TNF-’s effects and improved the levels of Akt and phosphorylated Akt. Because we showed that both the cleavage and ubiquitination of Akt are caspase dependent, it is not surprising that caspase inhibition improved the insulin response to a greater extent than proteasome inhibition. We repeated this experiment without CHX pretreatment and chose to also assess insulin-stimulated Mdm2 phosphorylation at Ser166, because Akt has been demonstrated to physically interact with Mdm2 and specifically phosphorylate it at that site (44, 45). When adipocytes were exposed to TNF- for 5 h and then stimulated with insulin for 30 min, the levels of Akt, Akt phosphorylated at Ser473, and phosphorylated Mdm2 were markedly decreased (Fig. 6B). Broad caspase inhibition attenuated this effect of TNF-. In contrast, MG132 did not improve phosphorylated Mdm2 and Akt levels. When the insoluble cellular fraction was analyzed by Western blot analysis, more Akt accumulated in the insoluble fraction when cells were preexposed to both MG132 and TNF- compared with only TNF- exposure (Fig. 6B, right panel). The protected Akt that accumulated in the insoluble cellular fraction did not appear to be phosphorylated in response to insulin (data not shown). Thus, although proteasome inhibition attenuated TNF--induced degradation of Akt, the protected Akt was probably unable to act upon Mdm2.
FIG. 6. Caspase and proteasome inhibition improves insulin-stimulated Akt phosphorylation, and caspase inhibition improves Akt-dependent insulin signaling in adipocytes preexposed to TNF-. A, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h, followed by preincubation with 50 μM BOC-D-FMK or 10 μM MG132 for 1 h before incubation with 3 nM TNF- for 5 h. Cells were washed with PBS (twice) and then treated for 30 min with 25 nM insulin. Cell lysates were prepared as usual and immunoblotted with anti-phospho-Akt (Ser473; anti-pAkt) and total anti-Akt. Immunoblotting with anti-p85 antibody was used as a loading control. B, 3T3-L1 adipocytes were preincubated with 50 μM BOC-D-FMK for 1 h before incubation with 3 nM TNF- for 5 h. Cells were washed with PBS (twice) and then treated for 30 min with 25 nM insulin. Cell lysates were prepared as usual and immunoblotted with anti-phospho-Akt (Ser473; anti-pAkt), total anti-Akt, and anti-phospho-Mdm2 (Ser166; pMdm2). Immunoblotting with anti-p85 antibody was used as a loading control. The insoluble fractions from cell lysates were sonicated in lysis buffer. Insoluble cellular fractions were resolved by 10% SDS-PAGE and then immunoblotted with anti-Akt antibody. Representative blots of at least three independent experiments are shown for A and B. C, Cells were treated as described in B, except that 100 nM insulin was used. 2-Deoxyglucose uptake was measured as described in Materials and Methods. The graph shows the mean ± SE of three independent experiments.
Short-term (4-h) exposure of 3T3-L1 adipocytes to TNF- was shown to inhibit insulin-stimulated glucose uptake (46). Therefore, we assessed whether caspase inhibition would improve insulin-stimulated glucose uptake by adipocytes exposed to TNF- for 5 h and then stimulated with insulin for 30 min. TNF- markedly inhibited insulin-stimulated glucose uptake by 42 ± 4%, and pretreatment with Boc-D-FMK improved uptake such that it was reduced by only 9 ± 1% (Fig. 6C). These data reinforce the concept that caspases act as significant mediators of impaired insulin signaling due to TNF-.
Discussion
TNF- induces multiple intracellular signals resulting in pleiotropic responses, such as the apoptosis of tumor cells or the regulation of inflammatory and immune responses. Although TNF- engages both TNF receptors 1 and 2, the former mediates most of its biological effects, including apoptosis and activation of nuclear factor-B. When TNF- binds TNF receptor 1, the intracellular domain of the receptor interacts with the adaptor protein TNF receptor-associated death domain, which recruits other adaptor proteins, including Fas-associated death domain (47). Fas-associated death domain recruits procaspase-8, which cleaves itself to become the initiator caspase-8 and, in turn, leads to activation of the executioner caspase-3, -6, and -7 (48). The executioner caspases cleave an array of cellular substrates that include structural proteins, proteins involved in DNA synthesis and repair, and proteins involved in signal transduction (49). TNF- is implicated as a mediator of sepsis- and obesity-related insulin resistance. Akt mediates several insulin-induced functions (15) and thereby plays a critical role in regulating metabolic homeostasis. In the present study, short-term exposure to TNF- decreased Akt protein levels in 3T3-L1 adipocytes. This effect was suppressed by caspase or proteasome inhibition. Attenuation of TNF--induced Akt degradation improved Akt-dependent insulin signaling in adipocytes.
A recent in vitro study demonstrated that incubation of Akt with active recombinant caspase-3, -6, or -7 resulted in the degradation of Akt and the generation of 44- and 40-kDa cleaved fragments (28). Akt1 was shown to contain two cleavage sites between the NH2-terminal pleckstrin homology domain and the kinase domain and an additional site in the COOH-terminal regulatory domain (28). We observed that TNF- induced the formation of a band in the 40–50 kDa range that immunoreacted with anti-Akt antibody and appeared as Akt protein levels were decreasing. Pretreatment with a general caspase inhibitor or a caspase-6 inhibitor before TNF- treatment potently attenuated the decline in Akt protein levels and suppressed the formation of that fragment. These results agree with reports that Akt degradation due to treatment with apoptosis inducers is caspase dependent (28, 50, 51). Akt1 was demonstrated to degrade over a period of 4–8 h after exposure to anti-Fas IgM antibodies or UV-C irradiation in Jurkat cells, and caspase inhibition or overexpression of Bcl-xL, which is an antiapoptotic protein that inhibits caspase activation, inhibited the effects of the anti-Fas antibody (50). Similarly, cells treated for several hours with concentrations of H2O2 that induce apoptosis exhibited caspase-dependent Akt degradation. However, Akt fragments could not be detected, and it was implied that other proteases probably acted downstream of the caspases to generate smaller peptide fragments of Akt (51). To our knowledge, ours is the first in vivo demonstration of a caspase-generated Akt fragment.
Proteins that have covalently attached to them several molecules of the 76-amino acid polypeptide called Ub are frequently targeted for degradation by the 26S proteasome (32). It was reported that cells treated with geldamycin, which binds the 90-kDa heat shock protein (Hsp90) ATP/adenosine diphosphate pocket such that it precludes folding of client proteins, results in the ubiquitination and proteasome-dependent degradation of Akt (43). Interestingly, growth factors were shown to stimulate both the activation and the proteasome-dependent degradation of Akt in vascular smooth muscle cells via the PI3 kinase pathway (52). Our study confirms that Akt can be ubiquitinated in vivo, and that an extracellular ligand-induced signal can lead to its ubiquitination and degradation via the proteasome. When 3T3-L1 adipocytes were exposed to TNF-, it induced the ubiquitination of Akt1 and Akt2. Pretreatment with proteasome inhibitors suppressed the degradation of both isoforms; this was observed regardless of whether adipocytes were preexposed to CHX before TNF- treatment. However, the use of CHX enabled detection of protected Akt in the soluble cellular fraction, whereas in its absence, it accumulated in the insoluble cellular fraction. This phenomenon resembles the reported effect of proteasome inhibition to cause Hsp90-associated Raf-1, receptor-interacting protein, and Akt accumulation in detergent-insoluble fractions; ubiquitination was induced with geldamycin, which interferes with Hsp90 function (41, 42, 43). Proteasome inhibition induces the formation of insoluble aggregates encaged by the intermediate filament protein vimentin; they contain misfolded proteins, 20S proteasomes, Ub, Hsp70, and Hsp90 (53). Because CHX disrupts vimentin filament organization (54), we speculate that CHX inhibited the formation of insoluble aggregates, which caused protected Akt to remain in the soluble cellular fraction.
Broad-spectrum caspase inhibition completely suppressed TNF--induced ubiquitination of Akt1 and Akt2, which demonstrates that caspase activation is required for that effect. We also determined that the executioner caspase-6 was a potent mediator of Akt1 ubiquitination and cleavage compared with other caspases. The role of executioner caspases in Akt1 cleavage is evident, because they are able to cleave Akt1 in vitro (28). Yet, how they mediated the ubiquitination of Akt1 and Akt2, and the signal that targets Akt1 and Akt2 for ubiquitination, are unclear. Because executioner caspases cleave a vast number of proteins, the possible mechanisms are numerous. The caspases may have cleaved and thereby inactivated a deubiquitinating enzyme or activated a latent E3 ligase that associates with Akt. Alternatively, because Hsp90 is a target of caspases during apoptosis, caspases could have cleaved Hsp90 that was associated with Akt, which would have impaired its function and resulted in Akt ubiquitination. The latter two possibilities are intriguing, because TNF- was observed to enhance the E3 ligase activity that was associated with Akt. We also must consider the possibility that the Akt fragments were ubiquitinated and comprised the ubiquitinated material that was immunoprecipitated with Akt. For example, caspase cleavage of Akt may have occurred first, followed by ubiquitination of the fragments and then degradation. This would explain the potent effect of caspase inhibition to suppress both the ubiquitination of Akt1 and the cleavage of Akt1. However, proteasome inhibition itself clearly and reproducibly attenuated the TNF--induced decline in full-length Akt1 and Akt2, which suggests that it is these full-length isoforms that are ubiquitinated and then degraded. Collectively, our results indicate that TNF- induces the caspase-dependent degradation of Akt via two distinct mechanisms: the cleavage of Akt1 and the ubiquitination of Akt1 and Akt2, which results in their degradation via the 26S proteasome (Fig. 7). Caspase- and proteosome-mediated Akt degradation was also recently demonstrated with endothelial cells deprived of vascular endothelial growth factor for 24 h. Furthermore, inhibiting the mammalian target of rapamycin when endothelial cells were exposed to vascular endothelial growth factor resulted in caspase induction and Akt ubiquitination; Akt degradation was prevented by a broad caspase or proteosome inhibitor (55). It would be interesting to test whether Akt ubiquitination in response to mammalian target of rapamycin inhibition in those endothelial cells is caspase dependent. In the present study, the significance of caspase- and proteasome-mediated degradation of Akt to the impairment of insulin signaling in adipocytes exposed to TNF- was demonstrated by the ability of caspase and proteasome inhibition to improve insulin-stimulated phosphorylation of Mdm2 at Ser166, which is phosphorylated by Akt at that site (44, 45). The ability of caspase inhibition to attenuate the inhibition of insulin-stimulated glucose uptake by TNF- underscores the role of caspases as important mediators of impaired insulin signaling.
FIG. 7. Schematic representation of Akt degradation due to TNF- exposure. TNF- induces caspase activation. Executioner caspases cleave Akt or mediate the ubiquitination of Akt by an unknown mechanism. Ubiquitinated Akt is then degraded by the 26S proteasome.
Hyperinsulinemia and elevated concentrations of TNF- influence adipocyte function in the metabolic syndrome associated with obesity and type II diabetes. The chronic cross-talk between prosurvival and proapoptotic signals impairs insulin responsiveness and sustains adipose tissue hypertrophy by weakening adipostat functions of TNF- such as apoptosis. Pathological concentrations of TNF- also impair adipocyte insulin signaling in sepsis and trauma. Insulin resistance in adipose tissue results in hyperlipidemia, which either initiates or exacerbates systemic insulin resistance (56, 57). We observed that short-term (<6 h) exposure of adipocytes to TNF- decreases Akt protein levels. In addition to Akt cleavage, investigation of this phenomenon revealed a novel mechanism by which TNF- can impair insulin signaling in adipocytes: the caspase-dependent ubiquitination of Akt1 and Akt2, which leads to their degradation via the 26S proteasome. We also demonstrated that both caspase and proteasome inhibition attenuated the TNF--induced degradation of Akt and improved Akt-dependent insulin signaling. Because sepsis, trauma, burns, obesity, and type II diabetes are replete with factors that can induce proapoptotic signaling in adipocytes, our findings probably extend beyond the effects of TNF-. TNF-, reactive oxidant species, nitric oxide, glucocorticoids, fatty acids, and leptin (58, 59, 60, 61, 62, 63) could synergize and potentiate the caspase activation that results in Akt degradation. Although the upstream pathways of apoptotic stimuli are diverse, their convergence at caspase activation may provide distinct targets for therapies aimed at improving the insulin sensitivity of adipocytes in hyperinflammatory states.
Acknowledgments
We thank Ingrid Wertz for technical advice.
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Abstract
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.
Introduction
THE PROINFLAMMATORY CYTOKINE TNF- is well known for its role in contributing to insulin resistance during infections, surgery, and trauma. The impaired ability of muscle and adipose tissue to respond to insulin frequently leads to hyperglycemia and hyperlipidemia in those inflammatory states. TNF- is also implicated as a mediator of insulin resistance in obesity and type 2 diabetes. Adipose tissue production of that cytokine is enhanced in insulin-resistant obese or diabetic animals and humans (1, 2), and administration of TNF- receptor IgG fusion protein or soluble TNF--binding protein, which neutralize TNF-, improved insulin responsiveness in obese and insulin-resistant animals (1, 3).
Several studies have demonstrated that TNF- impairs insulin responsiveness in cultured adipocytes. Characterization of the defect caused by TNF- has been elusive, because the cytokine interferes with insulin signaling at multiple levels. Although one study reported that acute exposure (15–120 min) of 3T3-L1 adipocytes to TNF- enhanced insulin-stimulated IRS-1 tyrosine phosphorylation and binding to phosphoinositide 3-kinase (PI3 kinase) (4), several demonstrated that acute treatment (<6 h) of human or 3T3-L1 adipocytes with TNF- decreased insulin-stimulated insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, PI3 kinase activity, and glucose transport (5, 6, 7, 8). The demonstrations that serine/threonine phosphorylation of IRS-1 impaired insulin signaling (9) and that TNF- induced serine/threonine phosphorylation of IRS-1 (10) suggest a mechanism for how the cytokine might attenuate IRS-1 function. Prolonged exposure to TNF- was shown to inhibit both IR autophosphorylation and IRS-1 phosphorylation (11). Chronic treatments also decreased transcript and protein levels of molecules that mediate the effects of insulin, including IRS-1, cyclic-nucleotide phosphodiesterase-3B (PDE3B), and glucose transporter-4 (12, 13); reduced amounts of these proteins resulted in impaired insulin responses. Short-term (1- to 6-h) treatment with TNF- also reduced Akt (also known as protein kinase B) protein levels in 3T3-L1 adipocytes (14), although the mechanism for its depletion or its effect on insulin action has not been explored.
Insulin triggers the autophosphorylation of intracellular tyrosine residues on the insulin receptor, which results in recruitment of IRS and activation of the lipid kinase PI3 kinase. PI3 kinase then generates 3'-phosphatidylinositol lipids that recruit Akt to the plasma membrane, where it is phosphorylated and thereby activated by 3'-phosphoinositide-dependent protein kinase-1 and -2. The Akt family consists of three highly homologous isoforms (Akt1, Akt2, and Akt3), and it mediates a number of insulin-induced functions, including glucose uptake, suppression of lipolysis via activation of PDE3B, and inhibition of glycogen synthase kinase-3, which enables activation of glycogen synthase (15). These responses are impaired in animals or humans with sepsis, obesity, and type 2 diabetes (16, 17, 18, 19, 20, 21), and TNF- has been shown to impair insulin-mediated glucose uptake in cultured adipocytes (5, 11, 12, 22). Because TNF- can decrease Akt expression in adipocytes, diminished Akt protein levels probably contribute to the impairment of insulin responses mediated by the PI3 kinase/Akt pathway in conditions noted for systemic inflammation.
In the present study we examined the mechanism by which TNF- decreases Akt protein levels in 3T3-L1 adipocytes. We report that TNF- decreases Akt expression via the caspase-dependent cleavage of Akt1 and the caspase-dependent ubiquitination of Akt1 and Akt2, which leads to their degradation by the 26S proteasome. We also demonstrate that inhibition of caspases or the 26S proteasome attenuates the effect of TNF- to impair Akt-dependent insulin signaling.
Materials and Methods
Reagents
TNF- was purchased from R&D Systems, Inc. (Minneapolis, MN). Cycloheximide (CHX), actinomycin (ACT), Ac-Leu-Leu-arginal (leupeptin), chloroquine, and N-acetyl-Leu-Leu-norleucinal were purchased from Sigma-Aldrich Corp. (St. Louis, MO). Z-Leu-Leu-Leu-CHO (MG132), t-butoxycarbonyl-Asp(O-Me)-fluoromethyl ketone (Boc-D-FMK), Z-Tyr-Val-Ala-Asp(OMe)-CH2F (Z-YVAD-FMK), Z-Val-Asp(OMe)-Val-Ala-Asp(OMe)-CH2F (Z-VDVAD-FMK), Z-DEVD-FMK, Z-Trp-Glu(OMe)-His-Asp(OMe)-CH2F (Z-WEHD-FMK), Z-Val-Glu(OMe)-Ile-Asp(OMe)-CH2F (Z-VEID-FMK), Z-Ile-Glu(OMe)-Thr-Asp(OMe)-CH2F (Z-IETD-FMK) and Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (Z-LEHD-FMK) were acquired from Calbiochem (San Diego, CA) and dissolved in Me2SO (Sigma-Aldrich Corp.) before their addition to the cell cultures. The antibodies used were polyclonal anti-Akt, polyclonal anti-phospho-Akt (Ser473), polyclonal anti-phospho-Mdm2 (Ser166), and anti-biotin, horseradish peroxidase-linked (Cell Signaling, Beverly, MA); polyclonal anti-Akt1/2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); polyclonal anti-Akt1, polyclonal anti-Akt2, polyclonal anti-Akt3, and polyclonal anti-p85 (PI3 kinase; Upstate Biotechnology, Lake Placid, NY); and monoclonal anti-ubiquitin (anti-Ub; Covance Research Products, Inc., Berkeley, CA).
3T3-L1 cell culture
3T3-L1 fibroblasts were grown at 37 C in 5% CO2 until 2 d post confluence in high glucose DMEM (Invitrogen Life Technologies, Inc., Grand Island, NY) supplemented with 10% calf serum (HyClone, Logan, UT), and then differentiation was induced by a 48-h incubation with DMEM supplemented with 10% fetal bovine serum (Invitrogen Life Technologies, Inc.), dexamethasone (0.25 mM; Sigma-Aldrich Corp.), methylisobutylxanthine (100 μM; Sigma-Aldrich Corp.), and insulin (10 μg/ml; Sigma-Aldrich Corp.). Cells continued to differentiate for another 5–9 d in DMEM supplemented with insulin (10 μg/ml) until more than 90% of the cells demonstrated the adipocyte morphology, as assessed by Oil Red O staining. Before treatment, cells were incubated in low glucose DMEM supplemented with 10% calf serum for 48 h, followed by incubation in low glucose DMEM supplemented with 0.5% calf serum for 24 h.
RNA extraction, RT, and real-time PCR
Total RNA was isolated from adipocytes with TRIzol (Invitrogen Life Technologies, Inc.) as previously described (23). Extracted RNA for each sample was quantified with RiboGreen (Molecular Probes, Eugene, OR), and 2 μg were reverse transcribed with SuperScript II Plus RNase H– Reverse Transcriptase using an oligo(deoxythymidine) primer (Invitrogen Life Technologies, Inc.). The LightCycler (Roche, Indianapolis, IN) was used for real-time PCR amplification. For real-time PCR, the following oligonucleotide primers were synthesized (Invitrogen Life Technologies, Inc.) using the cDNA sequences (GenBank sequence database of the National Center for Biotechnology Information) for murine Akt1 (M94335), Akt2 (U22445), and Akt3 (AF124142.1): 5'-GGCGTGGTCATGTACGAGATG-3' (Akt1 sense), 5'-TGAGCTGTGAACTCCTCATCGA-3' (Akt1 antisense), 5'-CATTCTTATGGAGGAGATCCGCTT-3' (Akt2 sense), 5'-GGTCCAGGCTGTCATATCGGT-3' (Akt2 antisense), 5'-AAGTATGACGACGACGGCATG-3' (Akt3 sense), and 5'-AGCAACAGCATGAGACCTTAGACTG-3' (Akt3 antisense). The sequences for the ?-actin oligonucleotide primers were previously reported (24). Real-time PCR efficiencies were calculated for each target gene (E = 10–1/slope), and all were determined to be approximately equal. Each reaction mixture consisted of 2.0 μl template DNA, 4 mM MgCl2, 0.5 μM of each primer, and LightCycler-DNA Master SYBR Green I in a total volume of 20 μl (Roche). The template DNA was diluted 50-fold before amplification with a protocol that consisted of 40 cycles with denaturation at 95 C for 1 sec, annealing at 56 C for 5 sec, and sequence extension at 72 C for 15 sec. A crossing point (Cp) for each sample was generated using the fit point method in the LightCycler software (Roche). Relative gene expression (over untreated cells) was obtained after normalization to ?-actin and determination of the difference between Cp in treated and untreated cells using the 2–Cp formula (25). Product identity was confirmed initially by sequence analysis and subsequently with the detection of a single product-specific melting temperature for each gene of interest with the LightCycler melting curve analysis program.
Immunoprecipitation and Western blotting
Lysates were extracted in solubilization buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 1 mM EGTA, 0.1% SDS, 5 mM N-ethylmalemide (Sigma-Aldrich Corp.), and protease and phosphatase inhibitor cocktail (Sigma-Aldrich Corp.). Lysates were cleared by centrifugation (16,000 x g) for 45 min, and protein concentrations were measured using a Bradford protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equivalent amounts of protein were resolved by 10% SDS-PAGE. For immunoprecipitations, 500 μg protein were incubated overnight at 4 C with the appropriate antibody, then precipitated the next day for 2 h with protein G (Upstate Biotechnology, Inc.). Immunoprecipitates were resolved by 5–15% SDS-PAGE. Resolved proteins were transferred onto a nitrocellulose membrane and then blocked for 1 h in Tris-buffered saline, pH 7.5, containing 0.5% Tween 20 and 5% milk. Membranes were incubated overnight at 4 C with the appropriate primary antibody. The next day, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h, and the immunoreactive bands were detected with an enhanced chemiluminescent reagent (Pierce Chemical Co., Rockford, IL). Images of the bands were photographed, and densitometry was performed with National Institutes of Health Image software.
In vitro ubiquitination assay
Immunoprecipitates were generated as described above, except that neither 0.1% SDS nor N-ethylmalemide was present in the lysis buffer. Beads loaded with Akt were washed with PBS, then incubated with 20 μl reaction mixture [300 mM HEPES (pH 7.2–7.6), 20 mM ATP, 50 mM MgCl2, and 2 mM dithiothreitol] and 1 μg biotin-N-terminal Ub (Boston Biochem, Inc., Cambridge, MA) that included either 40 ng E1 (Affinity Research, Ltd., Exeter, UK) and 600 ng each of UbcH1, UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH6, UbcH7, and UbcH10 (Affinity Research, Ltd.) or PBS. Samples were incubated and rocked for 1 h at 30 C. Reactions were stopped and incubated for 20 min with sodium dodecyl sulfate sample buffer containing 360 mM iodoacetamide, then analyzed by immunoblotting as described above.
Glucose uptake by 3T3-L1 adipocytes
Uptake of 2-deoxy-D-[3H]glucose (Amersham Biosciences, Arlington Heights, IL) was assessed in 3T3-L1 adipocytes differentiated in six-well plates as previously described (26) with minor modifications. After the treatments indicated in the figure legends, adipocytes were washed twice with 2 ml KRH buffer (129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.8 mM CaCl2, and 20 mM HEPES, pH 7.4), then incubated in 1 ml/well KRH buffer containing 200 nM insulin for 30 min at 37 C. Afterward, the cells were incubated in 1 ml KRH buffer containing 0.1 mM 2-deoxyglucose and 1 μCi/ml 2-deoxy-D-[3H]glucose for 5 min. Adipocytes were then washed three times with ice-cold PBS and solubilized in 1.0 ml 0.5 N NaOH and 0.1% sodium dodecyl sulfate. [3H]Glucose uptake was detected in 3 ml scintillant containing 0.3 ml lysate using a Beckman LS6500 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). Nonspecific deoxyglucose uptake (<10% of the total) was measured in the presence of 20 μM cytochalasin B and was subtracted from each determination to yield insulin-specific glucose uptake. Aliquots of the lysates were also subjected to protein concentration determination (Bio-Rad Laboratories).
Results
TNF- reduces Akt protein levels
3T3-L1 adipocytes were treated with TNF-, and the effects on Akt protein levels were characterized. Cell lysates were clarified, resolved by SDS-PAGE, and then immunoblotted with polyclonal anti-Akt. Incubation with TNF- for 5 h dose-dependently decreased Akt levels (Fig. 1A). TNF- also inhibited Akt protein levels in a time-dependent manner (Fig. 1B). TNF- did not affect the protein levels of the PI 3-kinase component p85. This is consistent with a previous report that TNF- treatment with a similar concentration and exposure period did not affect p85 protein levels in 3T3-L1 adipocytes (27). We therefore used p85 as a loading control for our subsequent studies. Immunoblotting with the polyclonal anti-Akt also revealed a low molecular mass immunoreactive band that migrated at approximately 44 kDa, which is in the predicted molecular mass range for a caspase-cleaved Akt fragment (40–50 kDa) (28).
FIG. 1. TNF- decreases Akt protein levels. A, Upper panel, Fully differentiated 3T3-L1 adipocytes were incubated with 0.06–6.0 nM TNF- for 5 h. Cell lysates were prepared in lysis buffer containing 1% Nonidet P-40. Lysates were clarified, resolved (25 μg/lane) by 10% SDS-PAGE gel, and then immunoblotted with a total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. Lower panel, The graph represents densitometry of Akt levels as a percentage of the control values in the blot shown (upper panel). A representative blot of at least three independent experiments is shown. B, Left panel, 3T3-L1 adipocytes were treated with 3 nM TNF- for 2–6 h. Cell lysates were resolved by 10% SDS-PAGE gel and immunoblotted with total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. Right panel, The graph represents densitometry of Akt levels as a percentage of the control values in the blot shown (left panel). A representative blot of at least three independent experiments is shown.
Reductions in Akt protein levels by TNF- involve caspases and the 26S proteasome
A recent study demonstrated by microarray analysis that exposure of 3T3-L1 adipocytes to TNF- for 24 h suppressed Akt2 (–3.6-fold), but not Akt1, gene expression (14). Using real-time RT-PCR, we observed that exposure to TNF- for 6 h suppressed mRNA levels of both Akt1 and Akt2 in 3T3-L1 adipocytes (Fig. 2A). To determine whether posttranslational effects were also involved in decreasing Akt levels during exposures to TNF-, adipocytes were pretreated with either the transcription inhibitor ACT or the translation inhibitor CHX before incubation with TNF-. Both ACT and CHX enhanced the TNF--induced decrease in Akt (Fig. 2B). Thus, the TNF--induced suppression of Akt protein levels in 3T3-L1 adipocytes involves both transcriptional and posttranslational mechanisms. To investigate the posttranslational effects of TNF- on Akt expression, subsequent experiments used CHX to control for transcriptional changes.
FIG. 2. TNF- decreases Akt1 and Akt2 mRNA levels and decreases Akt protein levels in the presence of a transcriptional or translational inhibitor with involvement of caspase(s) and the 26S proteasome. A, 3T3-L1 adipocytes were exposed to 3 nM TNF- for 3–6 h. Akt1, Akt2, and Akt3 mRNA levels were determined by real-time RT-PCR as described in Materials and Methods. B, 3T3-L1 adipocytes were preincubated with 10 μg/ml ACT or 0.5 mM CHX for 1 h, then exposed to 3 nM TNF- for 5 h. ACT and CHX were used as inhibitors of transcription and protein translation, respectively. Cell lysates were resolved by 10% SDS-PAGE gel and immunoblotted with a total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. A representative blot of at least three independent experiments is shown. C, Left panel, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h or with 10 μM MG132, 250 μM leupeptin, 50 μM BOC-D-FMK, or 150 μM chloroquine for 1 h, then exposed to 3 nM TNF- for 5 h. Cell lysates were prepared as described in Fig 1. Twenty-five micrograms of lysate were then resolved by 10% SDS-PAGE gel and immunoblotted with a total anti-Akt antibody. Immunoblotting with anti-p85 antibody was used as a loading control. Right panel, The graph represents densitometry of Akt levels in the blot shown in the left panel. A representative blot of at least three independent experiments is shown.
Posttranslational strategies employed by cells to decrease the levels of a given protein include proteasome-, calpain-, caspase-, and lysosome-mediated degradation. To test whether these pathways mediated the effect of TNF- to decrease Akt expression, cells were pretreated with specific inhibitors of these pathways (Fig. 2C). Pretreatment with the broad-spectrum caspase inhibitor Boc-D-FMK completely blocked the ability of TNF- to decrease Akt protein levels. A modest protection from a decrease in Akt levels was also observed with pretreatment with the 26S proteasome inhibitor, MG132 (29). However, the thiol protease inhibitor leupeptin (30) and the lysosomal inhibitor chloroquine (31) were unable to stabilize Akt levels. Boc-D-FMK also completely suppressed the TNF--induced generation of the approximately 44-kDa band. These data suggest that the observed low molecular bands generated in response to TNF- probably resulted from caspase-mediated Akt cleavage. Therefore, caspases and, to a lesser extent, the 26S proteasome are involved in the posttranslational down-regulation of Akt by TNF- in 3T3-L1 adipocytes.
Ubiquitinated Akt is increased by TNF- and suppressed by caspase inhibition
Proteins that have attached to them several molecules of the 76-amino acid polypeptide called Ub are efficiently targeted for degradation by the 26S proteasome (32). Because our data suggested proteasome involvement in Akt degradation due to TNF- exposure, we next determined whether TNF- increased the association of Ub with Akt. Akt was immunoprecipitated with polyclonal anti-Akt from lysates of adipocytes that were pretreated with CHX and then exposed to TNF- for up to 5 h. Immunoprecipitated protein was resolved by SDS-PAGE, then immunoblotted with a monoclonal antibody against Ub; the association of Ub with Akt results in the appearance of a high molecular weight protein ladder/smear. Ubiquitination of Akt was clearly increased after 5-h incubation with TNF- (Fig. 3A), and the levels of immunoprecipitated Akt were concomitantly decreased.
FIG. 3. TNF- induces caspase-dependent Akt ubiquitination. A, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h, then exposed to 3 nM TNF- for 0.5–5 h. Cell lysates were prepared as usual, except that 5 mM N-ethylmaleimide and 0.1% SDS were added to the lysis buffer. Cell lysates were immunoprecipitated with total anti-Akt antibody. Immunoprecipitates were resolved by 5–15% SDS-PAGE and immunoblotted with anti-Ub antibody (upper panel). The membrane was then stripped and immunoblotted with a total anti-Akt antibody (lower panel). B and C, Differentiated 3T3-L1 adipocytes were preincubated with 0.5 mM cycloheximide for 1 h, preincubated with 50 μM BOC-D-FMK or 10 μM MG132 for 1 h, then exposed to 3 nM TNF- for 5 h. Cell lysates were prepared as usual, except that 5 mM N-ethylmaleimide and 0.1% SDS were added to the lysis buffer. Cell lysates were immunoprecipitated with anti-Akt1 (B) or anti-Akt2 (C) antibody. Immunoprecipitates were resolved by 5–15% SDS-PAGE gel, then immunoblotted with an anti-Ub antibody (upper panel). The membrane was subsequently stripped and reprobed with an anti-Akt1 or anti-Akt2 antibody (lower panel). Representative blots of at least three independent experiments are shown for A, B, and C.
Recent studies indicate that the three isoforms of Akt can be differentially regulated by particular stimuli (33, 34, 35, 36). Adipocytes have been demonstrated to express more Akt2 than Akt1 and extremely little Akt3 (36, 37, 38, 39). Akt2 is required for insulin responsiveness in adipocytes, and Akt1 provides significant functional redundancy; the role of Akt3 appears insignificant (40). Our results heretofore do not indicate whether the two isoforms that are critical to insulin responsiveness are both decreased by TNF- or whether the caspase and Ub/proteasome pathways are involved in the degradation of both Akt1 and Akt2. Therefore, we sought to characterize how the two isoforms were affected by TNF-. Lysates from adipocytes pretreated with CHX and then exposed to TNF- for 5 h were subjected to immunoprecipitation with isoform-specific antibodies and subsequent Western blot analysis with anti-Ub or anti-Akt. TNF- increased the ubiquitination of Akt1 and Akt2 (Fig. 3, B and C, upper panels) and decreased levels of both isoforms (Fig. 3, B and C, lower panels). Although the low molecular weight fragment was detected with only Akt1 immunoprecipitation, we are presently unable to rule out the possibility that caspases also cleaved Akt2. Broad caspase inhibition markedly decreased the accumulation of ubiquitinated Akt1 and Akt2 due to TNF- exposure, stabilized the two isoforms, and prevented accumulation of the Akt1 fragment. Proteasome inhibition attenuated the decline in Akt1 levels due to TNF- exposure and protected Akt2. Thus, TNF- regulates Akt1 and Akt2 in a similar manner by inducing the caspase-dependent ubiquitination and 26S proteasome-mediated degradation of both isoforms.
Caspase-6 is a key mediator for TNF--induced ubiquitination and cleavage of Akt1
Because broad caspase inhibition prevented the TNF--induced degradation and ubiquitination of Akt1 and Akt2, we tested the involvement of particular initiator, effector, and inflammatory caspases. Our data clearly showed that Akt1 was cleaved by caspases (Fig. 3B); thus, the following experiments were designed to detect Akt1 cleavage and ubiquitination. Adipocytes were pretreated with CHX and a panel of caspase inhibitors, then exposed to TNF-. Lysates were subjected to immunoprecipitation with polyclonal anti-Akt1 and subsequent Western blot analysis. As previously demonstrated, the broad-spectrum caspase inhibitor Boc-D-FMK completely inhibited Akt1 ubiquitination and the decline in Akt1 protein levels (Fig. 4A, upper and lower panels, respectively, lane B). The caspase-6 inhibitor, Z-VEID-FMK, inhibited both TNF--induced Akt1 ubiquitination (Fig. 4A, upper panel, and Fig. 4B, lane 6) and degradation (Fig 4A, lower panel, lane 6) to nearly the same extent as the broad-spectrum caspase inhibitor. None of the other caspase inhibitors was able to suppress both TNF--induced Akt1 ubiquitination and degradation, although caspase-8 inhibition appears to have reduced Akt1 ubiquitination. These results indicate that caspase-6 is probably a potent mediator of TNF- to increase Akt1 ubiquitination and cleave Akt1.
FIG. 4. TNF- induced Akt ubiquitination and degradation are attenuated by caspase-6 inhibition. A, Before TNF- exposure for 5 h, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h and preincubated for 1 h with 50 μM of the broad-spectrum caspase inhibitor BOC-D-FMK (lane B), 50 μM of the caspase-1 inhibitor Z-YVAD-FMK (lane 1), 50 μM of the caspase-2 inhibitor Z-VDVAD-FMK (lane 2), 50 μM of the caspase-3 inhibitor Z-DEVD-FMK (lane 3), 50 μM of the caspase-5 inhibitor Z-WEHD-FMK (lane 5), 50 μM of the caspase-6 inhibitor Z-VEID-FMK (lane 6), 50 μM of the caspase-8 inhibitor Z-IETD-FMK (lane 8), or 50 μM of the caspase-9 inhibitor Z-LEHD-FMK (lane 9). Cell lysates were prepared as usual, except that 5 mM N-ethylmaleimide and 0.5% SDS were added to the lysis buffer. Cell lysates were immunoprecipitated with anti-Akt1 antibody. Immunoprecipitates were resolved by 5–15% SDS-PAGE gel, then immunoblotted with an anti-Ub antibody. The membrane was subsequently stripped and reprobed with an anti-Akt1 antibody. B, Densitometric analysis for levels of ubiquitinated Akt1 (Ub-Akt1). A representative blot of at least two independent experiments is shown.
We verified the physiological relevance of the observed posttranslational effects of TNF- by examining the roles of caspases and the 26S proteasome in reducing Akt expression in the absence of CHX. The results were similar, except that proteasome inhibition caused protected Akt to accumulate in the insoluble cellular fraction (data not shown). This phenomenon resembles the reported effect of proteasome inhibition to cause ubiquitinated Raf-1, receptor-interacting protein, and Akt accumulation in detergent-insoluble fractions (41, 42, 43).
TNF- increases the E3 ligase activity associated with Akt
We next examined the role of TNF- in the induction of the Ub machinery. Ub is first activated via ATP-dependent formation of a thiol ester bond with a cysteine on a Ub-activating enzyme (E1), transferred to a cysteine on a Ub-conjugating enzyme (E2), and then it is covalently attached to a lysine on the protein to be degraded in a reaction catalyzed by a Ub-protein ligase (E3) (32). We hypothesized that TNF- induces the ubiquitination of Akt by enhancing the association of Akt with an E3 ligase. To test this possibility, lysates from cells incubated with TNF- for 1.5 or 3.0 h were immunoprecipitated with polyclonal anti-Akt, and the resulting immunocomplexes were incubated with biotinylated Ub and ATP and with or without E1 and E2 for 1 h. The reaction mixtures were resolved by SDS-PAGE and then immunoblotted with anti-biotin antibody or monoclonal anti-Akt1. Reaction mixtures derived from 3T3-L1 adipocytes that were incubated with TNF- for 1.5 h, but not 3.0 h, demonstrated an increased ability, compared with those from untreated cells, to generate Akt that was associated with biotinylated Ub (Fig. 5). The result at 3.0 h is probably due to the decreased amount of Akt that was immunoprecipitated from the lysate of adipocytes exposed to TNF-. Therefore, because the levels of immunoprecipitated Akt were the same for both TNF--treated (1.5 h) and untreated cells, these data indicate that TNF- enhanced the association of an E3 ligase with Akt. As expected, these data also demonstrate the requirement for E2, E1, and ATP for the in vitro ubiquitination reaction.
FIG. 5. TNF- enhances the E3 ligase activity associated with Akt. 3T3-L1 adipocytes were incubated with 3 nM TNF- for 1.5 or 3 h. Cell lysates were prepared as usual. Cell lysates (500 μg) were immunoprecipitated with total anti-Akt antibody. Beads loaded with Akt were incubated with rocking for 1 h at 30 C in a reaction mixture that included ATP, E1, and E2 or ATP and PBS (see Materials and Methods for details). Immunoprecipitates were resolved by 5–15% SDS-PAGE and immunoblotted with -biotin antibody. The membrane was subsequently stripped and reprobed with an anti-Akt1 antibody. Representative blots of at least three independent experiments are shown.
Caspase inhibition improves insulin signaling via Akt in adipocytes exposed to TNF-
TNF- is implicated as a mediator of sepsis- and obesity-related insulin resistance. Therefore, we tested whether diminished Akt levels due to TNF- exposure would impair Akt-dependent signaling. When adipocytes were pretreated with CHX, exposed to TNF- for another 5 h, and then stimulated with insulin for 30 min, the levels of Akt and phosphorylated Akt were markedly suppressed (Fig. 6A). Both Boc-D-FMK and MG132 impaired TNF-’s effects and improved the levels of Akt and phosphorylated Akt. Because we showed that both the cleavage and ubiquitination of Akt are caspase dependent, it is not surprising that caspase inhibition improved the insulin response to a greater extent than proteasome inhibition. We repeated this experiment without CHX pretreatment and chose to also assess insulin-stimulated Mdm2 phosphorylation at Ser166, because Akt has been demonstrated to physically interact with Mdm2 and specifically phosphorylate it at that site (44, 45). When adipocytes were exposed to TNF- for 5 h and then stimulated with insulin for 30 min, the levels of Akt, Akt phosphorylated at Ser473, and phosphorylated Mdm2 were markedly decreased (Fig. 6B). Broad caspase inhibition attenuated this effect of TNF-. In contrast, MG132 did not improve phosphorylated Mdm2 and Akt levels. When the insoluble cellular fraction was analyzed by Western blot analysis, more Akt accumulated in the insoluble fraction when cells were preexposed to both MG132 and TNF- compared with only TNF- exposure (Fig. 6B, right panel). The protected Akt that accumulated in the insoluble cellular fraction did not appear to be phosphorylated in response to insulin (data not shown). Thus, although proteasome inhibition attenuated TNF--induced degradation of Akt, the protected Akt was probably unable to act upon Mdm2.
FIG. 6. Caspase and proteasome inhibition improves insulin-stimulated Akt phosphorylation, and caspase inhibition improves Akt-dependent insulin signaling in adipocytes preexposed to TNF-. A, 3T3-L1 adipocytes were preincubated with 0.5 mM CHX for 1 h, followed by preincubation with 50 μM BOC-D-FMK or 10 μM MG132 for 1 h before incubation with 3 nM TNF- for 5 h. Cells were washed with PBS (twice) and then treated for 30 min with 25 nM insulin. Cell lysates were prepared as usual and immunoblotted with anti-phospho-Akt (Ser473; anti-pAkt) and total anti-Akt. Immunoblotting with anti-p85 antibody was used as a loading control. B, 3T3-L1 adipocytes were preincubated with 50 μM BOC-D-FMK for 1 h before incubation with 3 nM TNF- for 5 h. Cells were washed with PBS (twice) and then treated for 30 min with 25 nM insulin. Cell lysates were prepared as usual and immunoblotted with anti-phospho-Akt (Ser473; anti-pAkt), total anti-Akt, and anti-phospho-Mdm2 (Ser166; pMdm2). Immunoblotting with anti-p85 antibody was used as a loading control. The insoluble fractions from cell lysates were sonicated in lysis buffer. Insoluble cellular fractions were resolved by 10% SDS-PAGE and then immunoblotted with anti-Akt antibody. Representative blots of at least three independent experiments are shown for A and B. C, Cells were treated as described in B, except that 100 nM insulin was used. 2-Deoxyglucose uptake was measured as described in Materials and Methods. The graph shows the mean ± SE of three independent experiments.
Short-term (4-h) exposure of 3T3-L1 adipocytes to TNF- was shown to inhibit insulin-stimulated glucose uptake (46). Therefore, we assessed whether caspase inhibition would improve insulin-stimulated glucose uptake by adipocytes exposed to TNF- for 5 h and then stimulated with insulin for 30 min. TNF- markedly inhibited insulin-stimulated glucose uptake by 42 ± 4%, and pretreatment with Boc-D-FMK improved uptake such that it was reduced by only 9 ± 1% (Fig. 6C). These data reinforce the concept that caspases act as significant mediators of impaired insulin signaling due to TNF-.
Discussion
TNF- induces multiple intracellular signals resulting in pleiotropic responses, such as the apoptosis of tumor cells or the regulation of inflammatory and immune responses. Although TNF- engages both TNF receptors 1 and 2, the former mediates most of its biological effects, including apoptosis and activation of nuclear factor-B. When TNF- binds TNF receptor 1, the intracellular domain of the receptor interacts with the adaptor protein TNF receptor-associated death domain, which recruits other adaptor proteins, including Fas-associated death domain (47). Fas-associated death domain recruits procaspase-8, which cleaves itself to become the initiator caspase-8 and, in turn, leads to activation of the executioner caspase-3, -6, and -7 (48). The executioner caspases cleave an array of cellular substrates that include structural proteins, proteins involved in DNA synthesis and repair, and proteins involved in signal transduction (49). TNF- is implicated as a mediator of sepsis- and obesity-related insulin resistance. Akt mediates several insulin-induced functions (15) and thereby plays a critical role in regulating metabolic homeostasis. In the present study, short-term exposure to TNF- decreased Akt protein levels in 3T3-L1 adipocytes. This effect was suppressed by caspase or proteasome inhibition. Attenuation of TNF--induced Akt degradation improved Akt-dependent insulin signaling in adipocytes.
A recent in vitro study demonstrated that incubation of Akt with active recombinant caspase-3, -6, or -7 resulted in the degradation of Akt and the generation of 44- and 40-kDa cleaved fragments (28). Akt1 was shown to contain two cleavage sites between the NH2-terminal pleckstrin homology domain and the kinase domain and an additional site in the COOH-terminal regulatory domain (28). We observed that TNF- induced the formation of a band in the 40–50 kDa range that immunoreacted with anti-Akt antibody and appeared as Akt protein levels were decreasing. Pretreatment with a general caspase inhibitor or a caspase-6 inhibitor before TNF- treatment potently attenuated the decline in Akt protein levels and suppressed the formation of that fragment. These results agree with reports that Akt degradation due to treatment with apoptosis inducers is caspase dependent (28, 50, 51). Akt1 was demonstrated to degrade over a period of 4–8 h after exposure to anti-Fas IgM antibodies or UV-C irradiation in Jurkat cells, and caspase inhibition or overexpression of Bcl-xL, which is an antiapoptotic protein that inhibits caspase activation, inhibited the effects of the anti-Fas antibody (50). Similarly, cells treated for several hours with concentrations of H2O2 that induce apoptosis exhibited caspase-dependent Akt degradation. However, Akt fragments could not be detected, and it was implied that other proteases probably acted downstream of the caspases to generate smaller peptide fragments of Akt (51). To our knowledge, ours is the first in vivo demonstration of a caspase-generated Akt fragment.
Proteins that have covalently attached to them several molecules of the 76-amino acid polypeptide called Ub are frequently targeted for degradation by the 26S proteasome (32). It was reported that cells treated with geldamycin, which binds the 90-kDa heat shock protein (Hsp90) ATP/adenosine diphosphate pocket such that it precludes folding of client proteins, results in the ubiquitination and proteasome-dependent degradation of Akt (43). Interestingly, growth factors were shown to stimulate both the activation and the proteasome-dependent degradation of Akt in vascular smooth muscle cells via the PI3 kinase pathway (52). Our study confirms that Akt can be ubiquitinated in vivo, and that an extracellular ligand-induced signal can lead to its ubiquitination and degradation via the proteasome. When 3T3-L1 adipocytes were exposed to TNF-, it induced the ubiquitination of Akt1 and Akt2. Pretreatment with proteasome inhibitors suppressed the degradation of both isoforms; this was observed regardless of whether adipocytes were preexposed to CHX before TNF- treatment. However, the use of CHX enabled detection of protected Akt in the soluble cellular fraction, whereas in its absence, it accumulated in the insoluble cellular fraction. This phenomenon resembles the reported effect of proteasome inhibition to cause Hsp90-associated Raf-1, receptor-interacting protein, and Akt accumulation in detergent-insoluble fractions; ubiquitination was induced with geldamycin, which interferes with Hsp90 function (41, 42, 43). Proteasome inhibition induces the formation of insoluble aggregates encaged by the intermediate filament protein vimentin; they contain misfolded proteins, 20S proteasomes, Ub, Hsp70, and Hsp90 (53). Because CHX disrupts vimentin filament organization (54), we speculate that CHX inhibited the formation of insoluble aggregates, which caused protected Akt to remain in the soluble cellular fraction.
Broad-spectrum caspase inhibition completely suppressed TNF--induced ubiquitination of Akt1 and Akt2, which demonstrates that caspase activation is required for that effect. We also determined that the executioner caspase-6 was a potent mediator of Akt1 ubiquitination and cleavage compared with other caspases. The role of executioner caspases in Akt1 cleavage is evident, because they are able to cleave Akt1 in vitro (28). Yet, how they mediated the ubiquitination of Akt1 and Akt2, and the signal that targets Akt1 and Akt2 for ubiquitination, are unclear. Because executioner caspases cleave a vast number of proteins, the possible mechanisms are numerous. The caspases may have cleaved and thereby inactivated a deubiquitinating enzyme or activated a latent E3 ligase that associates with Akt. Alternatively, because Hsp90 is a target of caspases during apoptosis, caspases could have cleaved Hsp90 that was associated with Akt, which would have impaired its function and resulted in Akt ubiquitination. The latter two possibilities are intriguing, because TNF- was observed to enhance the E3 ligase activity that was associated with Akt. We also must consider the possibility that the Akt fragments were ubiquitinated and comprised the ubiquitinated material that was immunoprecipitated with Akt. For example, caspase cleavage of Akt may have occurred first, followed by ubiquitination of the fragments and then degradation. This would explain the potent effect of caspase inhibition to suppress both the ubiquitination of Akt1 and the cleavage of Akt1. However, proteasome inhibition itself clearly and reproducibly attenuated the TNF--induced decline in full-length Akt1 and Akt2, which suggests that it is these full-length isoforms that are ubiquitinated and then degraded. Collectively, our results indicate that TNF- induces the caspase-dependent degradation of Akt via two distinct mechanisms: the cleavage of Akt1 and the ubiquitination of Akt1 and Akt2, which results in their degradation via the 26S proteasome (Fig. 7). Caspase- and proteosome-mediated Akt degradation was also recently demonstrated with endothelial cells deprived of vascular endothelial growth factor for 24 h. Furthermore, inhibiting the mammalian target of rapamycin when endothelial cells were exposed to vascular endothelial growth factor resulted in caspase induction and Akt ubiquitination; Akt degradation was prevented by a broad caspase or proteosome inhibitor (55). It would be interesting to test whether Akt ubiquitination in response to mammalian target of rapamycin inhibition in those endothelial cells is caspase dependent. In the present study, the significance of caspase- and proteasome-mediated degradation of Akt to the impairment of insulin signaling in adipocytes exposed to TNF- was demonstrated by the ability of caspase and proteasome inhibition to improve insulin-stimulated phosphorylation of Mdm2 at Ser166, which is phosphorylated by Akt at that site (44, 45). The ability of caspase inhibition to attenuate the inhibition of insulin-stimulated glucose uptake by TNF- underscores the role of caspases as important mediators of impaired insulin signaling.
FIG. 7. Schematic representation of Akt degradation due to TNF- exposure. TNF- induces caspase activation. Executioner caspases cleave Akt or mediate the ubiquitination of Akt by an unknown mechanism. Ubiquitinated Akt is then degraded by the 26S proteasome.
Hyperinsulinemia and elevated concentrations of TNF- influence adipocyte function in the metabolic syndrome associated with obesity and type II diabetes. The chronic cross-talk between prosurvival and proapoptotic signals impairs insulin responsiveness and sustains adipose tissue hypertrophy by weakening adipostat functions of TNF- such as apoptosis. Pathological concentrations of TNF- also impair adipocyte insulin signaling in sepsis and trauma. Insulin resistance in adipose tissue results in hyperlipidemia, which either initiates or exacerbates systemic insulin resistance (56, 57). We observed that short-term (<6 h) exposure of adipocytes to TNF- decreases Akt protein levels. In addition to Akt cleavage, investigation of this phenomenon revealed a novel mechanism by which TNF- can impair insulin signaling in adipocytes: the caspase-dependent ubiquitination of Akt1 and Akt2, which leads to their degradation via the 26S proteasome. We also demonstrated that both caspase and proteasome inhibition attenuated the TNF--induced degradation of Akt and improved Akt-dependent insulin signaling. Because sepsis, trauma, burns, obesity, and type II diabetes are replete with factors that can induce proapoptotic signaling in adipocytes, our findings probably extend beyond the effects of TNF-. TNF-, reactive oxidant species, nitric oxide, glucocorticoids, fatty acids, and leptin (58, 59, 60, 61, 62, 63) could synergize and potentiate the caspase activation that results in Akt degradation. Although the upstream pathways of apoptotic stimuli are diverse, their convergence at caspase activation may provide distinct targets for therapies aimed at improving the insulin sensitivity of adipocytes in hyperinflammatory states.
Acknowledgments
We thank Ingrid Wertz for technical advice.
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