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Endocrinology Vol. 141, No. 7 2508-2517
Copyright © 2000 by The Endocrine Society

ARTICLES

Insulin Inhibits the Ubiquitin-Dependent Degrading Activity of the 26S Proteasome1

Robert G. Bennett, Frederick G. Hamel and William C. Duckworth2

Veterans Affairs Medical Center, and the Departments of Internal Medicine and Biochemistry and Molecular Biology (R.G.B., W.C.D.) and Pharmacology (F.G.H.), University of Nebraska Medical Center, Omaha, Nebraska 68198-3020

Address all correspondence and requests for reprints to: Robert G. Bennett, Ph.D., Research Service (151), Veterans Affairs Medical Center, 4101 Woolworth Avenue, Omaha, Nebraska 68105. E-mail: rgbennet{at}unmc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A major metabolic effect of insulin is inhibition of cellular proteolysis, but the proteolytic systems involved are unclear. Tissues have multiple proteolytic systems, including the ATP- and ubiquitin-dependent proteasome pathway. The effect of insulin on this pathway was examined in vitro and in cultured cells. Insulin inhibited ATP- and ubiquitin-dependent lysozyme degradation more than 90% by reticulocyte extract, in a dose-dependent manner (IC50 ~ 50 nM). Insulin did not reduce the conjugation of ubiquitin to lysozyme and was not itself ubiquitin-conjugated. In HepG2 cells, insulin increased ubiquitin-conjugate accumulation 80%. The association between the 26S proteasome and an intracellular protease, the insulin-degrading enzyme (IDE), was examined by a purification scheme designed to enrich for the 26S proteasome. Copurification of IDE activity and immunoreactivity with the proteasome were detected through several chromatographic steps. Glycerol gradient analysis revealed cosedimentation of IDE with the 20S proteasome and possibly with the 26S proteasome. The proteasome-associated IDE was displaced when the samples were treated with insulin. These results suggest that insulin regulates protein catabolism, at least in part, by decreasing ubiquitin-mediated proteasomal activity, and provides a new target for insulin action. The displacement of IDE from the proteasome provides a mechanism for this insulin action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN PROMOTES CELLULAR growth and maintenance by a wide variety of both anabolic and anticatabolic actions, including the inhibition of overall proteolysis (1). Although insulin administration causes an increase in the synthesis of some specific proteins, the effect of insulin on the level of total cellular protein is attributable almost entirely to decreased protein degradation (2, 3). However, the proteolytic systems regulated by insulin are unclear. In a variety of cell types, the absence of insulin and amino acids induces formation of autophagic vacuoles and subsequent lysosomal degradation, which is reversible by the addition of insulin (2, 4, 5). Little is known about how insulin regulates proteolytic rates under more physiological conditions, such as postprandial decreases in protein degradation. Recent studies suggest that nonlysosomal mechanisms may be involved (6).

The proteasome has been identified as the mechanism responsible for the majority of total cellular proteolysis (7). The proteasome is involved in the degradation of short- and long-lived proteins, the removal of damaged proteins, cell cycle control, transcription factor activation, and antigen presentation (8). Non-ATP-dependent peptide and protein degradation is carried out by the 20S proteasome, the catalytic core of all proteasome forms (9). The major nonlysosomal ATP-dependent proteolytic pathway is the ubiquitin system (10), involving the 26S proteasome, consisting of the 20S enzyme and regulatory subunits. The ubiquitin pathway requires both ATP and conjugation of a small polypeptide, ubiquitin (Ub), to lysine residues of target substrates (11, 12). This ligation of ubiquitin by a series of ubiquitin-conjugating enzymes is repeated, to produce polyubiquitin chains, which serve as targeting signals for degradation of the protein by the 26S proteasome. The importance of the ubiquitin-proteasome pathway of protein degradation is becoming increasingly apparent as more endogenous protein substrates are identified and regulation of the pathway in a number of pathophysiological states, including starvation and sepsis, is observed (13, 14). In muscle from diabetic rats, insulin reversed increased ATP-dependent proteolysis (15), but the effect of insulin on the ubiquitin pathway of ATP-dependent proteolysis is, as yet, unclear.

Previously, we showed that insulin inhibits ATP-independent peptide-degrading activity by the proteasome in vitro (16, 17) and in cultured cells (18). The ability of insulin to inhibit this proteasome activity was mediated by a copurifying protein, the insulin-degrading enzyme (IDE) (19, 20, 21). The IDE is a primarily cytosolic enzyme that is the initial member of a family of zinc metalloproteinases (22), and it is responsible for the degradation of insulin in most tissues (23, 24). In the present study, we found that insulin inhibited ATP- and ubiquitin-dependent proteolysis in an in vitro extract, and that insulin increased the accumulation of ubiquitin-conjugated proteins in cultured hepatoma cells. Furthermore, we observed copurification of IDE through several steps of a skeletal muscle 26S proteasome purification series. Insulin treatment resulted in dissociation of IDE from the proteasome. These data support involvement of IDE in the regulation of proteasome activity by insulin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Human crystalline insulin and 125I-insulin were generously provided by Ronald Chance and Bruce Frank, respectively (Lilly Research Laboratories, Indianapolis, IN). The C59 antibody specific for ubiquitin was provided by Terrence Donohue (Omaha Veterans Affairs Medical Center) The IDE-specific monoclonal antibody 9B12 was provided by Richard Roth (Stanford University, Stanford, CA) (25). The polyclonal antiserum against the 20S proteasome was provided by Walter Ward (University of Texas Health Science Center, San Antonio, TX) (26). Curve-fitting and statistical analyses were performed using GraphPad Prism version 3.00 (GraphPad Software, Inc., San Diego, CA).

Ub-conjugation and Ub-dependent degradation of lysozyme
Rabbit reticulocyte lysate (Green Hectares, Oregon, WI) was fractionated to produce Fraction II, as described (27). Lysozyme and ubiquitin were iodinated by the chloramine-T method (28). Ub-dependent degradation of 125I-lysozyme by Fraction II (50 µg total protein) was measured by trichloroacetic acid solubility ,as described (29), except that dithiothreitol was limited to that present in the enzyme, to minimize disulfide reduction of insulin. The reaction included either ATP-regenerating (22) or ATP-depleting (60 U/ml hexokinase, 10 mM 2-deoxyglucose) systems, and the rate of ATP-dependent degradation was taken as the difference. The total time of incubation was 3 h at 37 C, which typically resulted in approximately 10% trichloroacetic acid solubility in the presence of both ATP and ubiquitin, with approximately 1% solubility in the absence of ATP and/or ubiquitin. Some experiments were performed with the addition of 50 µg/ml of the inhibitory monoclonal anti-IDE antibody C20–3.1A (17, 19). Unlabeled lysozyme was conjugated to 125I-ubiquitin using Fraction II with hemin to prevent breakdown of the ubiquitin-lysozyme conjugates (22). Insulin-ubiquitin conjugation was assessed as above, except that 125I-labeled insulin was used instead of 125I-ubiquitin, with 1 mg/ml bacitracin to reduce the amount of insulin degradation. The levels of 125I-ubiquitin-protein conjugation were analyzed by SDS-PAGE and PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA).

Immunodetection of ubiquitin-protein conjugates from HepG2 cell lysates
Subconfluent cultures of human hepatoma (HepG2) cells (American Type Culture Collection, Rockville, MD) were maintained in DMEM with 10% FBS, then changed to serum-free DMEM for 16 h before treatment with insulin for 2 h. Cells were washed with ice-cold PBS, then scraped into lysis buffer (50 mM Tris (pH 7.4), containing 1% Triton X-100; 150 mM NaCl; 5 mM EDTA; 5 mM N-ethylmaleimide; 1 mM Na3VO4; 40 mM NaF; 1 mM phenylmethyl sulfonyl fluoride; 10 µg/ml each leupeptin, aprotinin, and soybean trypsin inhibitor; 10 µM pepstatin-A; 10 µM dichloroisocoumarin; and 10 µM hemin). After one freeze-thaw cycle, the samples were centrifuged at 10,000 x g for 10 min, and the supernatants were assayed for protein content by bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL). Samples were resolved on 12% SDS-PAGE, transferred to Immobilon-P (Millipore Corp., Bedford, MA), then probed with C59 antibody specific for ubiquitin (30), and visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Loading controls were performed by reprobing with anti {alpha}-tubulin monoclonal antibody (Sigma, St. Louis, MO) and alkaline phosphatase detection. Immunoreactive proteins, were quantitated by densitometry.

Purification of 26S proteasome from rat skeletal muscle
The 26S proteasome was prepared from rat skeletal muscle by DEAE-Sephacel chromatography, polyethylene glycol precipitation, phosphocellulose chromatography, and centrifugation at 100,00 x g, as described earlier (31) but with the omission of dithiothreitol and EDTA from the buffers to preserve IDE-proteasome interaction. Further analysis was performed on 10–40% linear glycerol gradients in 50 mM Tris (pH 7.5), containing 0.1 mM ATP (30 ml total). The gradients were centrifuged at 100,000 x g for 16 h, then fractions (0.9 ml) were collected from the bottom. Aliquots of each fraction were assayed for insulin-degrading activity and for succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (LLVY)-degrading activity, with and without 0.02% SDS (17). Samples were analyzed for IDE or the proteasome by Western blotting and detection by enhanced chemiluminescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Because insulin was previously shown to inhibit the ATP-independent activity of the proteasome, we sought to determine the effect of insulin on ATP- and ubiquitin-dependent proteolysis. A partially purified preparation from ATP-depleted rabbit reticulocyte lysate, known as Fraction II (27), is largely devoid of endogenous ATP and ubiquitin but contains the necessary factors for both the formation and degradation of ubiquitin-protein conjugates. The effect of insulin on ATP-dependent degradation of 125I-lysozyme in this system was examined (Fig. 1Go). Insulin inhibited ATP-dependent degradation of 125I-lysozyme in a dose-dependent manner, with an apparent IC50 of approximately 50 nM, and with maximal inhibition approximately 92%. Under the same conditions, insulin degradation (dotted line) was inhibited with a similar curve (IC50 ~60 nM), consistent with the action of IDE. To further investigate the role of IDE in the ubiquitin pathway, the effect of insulin on 125I-lysozyme degradation in the same system as above was determined in the presence and absence of an inhibitory monoclonal antibody directed against IDE (17, 19). In the presence of the antibody, insulin had no effect on ubiquitin- and ATP-dependent activity of the proteasome (Fig. 1BGo), suggesting that IDE is involved in insulin regulation of this pathway.



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  		Figure 1. Insulin inhibits ATP- and ubiquitin-dependent degradation of 125I-lysozyme in reticulocyte lysate extract. The ATP- and ubiquitin-dependent degradation of 125I-lysozyme in rabbit reticulocyte lysate Fraction II (27 ) was measured by trichloroacetic acid solubility, as described in Materials and Methods. Data are expressed as percent of maximal activity, mean ± SEM for three independent experiments (*, P < 0.05; **, P < 0.01). A, Solid line and symbols: dose response of insulin inhibition of ATP-dependent degradation of 125I-lysozyme; dotted line: degradation of 125I-insulin under the same conditions. B, Effect of the inhibitory anti-IDE antibody C20–3.1A on insulin inhibition of ATP-dependent degradation of 125I-lysozyme.

 
Because the ubiquitin pathway of proteolysis requires two separate steps (ubiquitin-conjugation and subsequent degradation), we sought to determine whether insulin decreased the conjugation of ubiquitin to proteins. The same in vitro extract system was used, with the addition of hemin, a potent inhibitor of the proteasome that allows quantification of ubiquitin conjugation (32). The effect of insulin on the conjugation of 125I-ubiquitin to unlabeled lysozyme (as detected by the shift in the migration of 125I-Ub to higher molecular weight species) is shown in the autoradiogram in Fig. 2Go. Insulin had no effect on ubiquitin conjugation to lysozyme (compare lanes 1 and 3). The addition of excess unlabeled ubiquitin as a competitor (lanes 2 and 4) reduced the levels of radioactive conjugates nearly to the level present in the absence of ATP (lane 5).



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  		Figure 2. Insulin has no effect on the conjugation of 125I-ubiquitin to lysozyme. Conjugation of 125I-Ub to lysozyme was performed as described in Materials and Methods, with and without 1 µM insulin. A, Equal radioactivity (15 kcpm) was applied to 12% SDS-PAGE. The migration of 125I- Ub standard (lane 6) is shown (arrow). A contaminant present during the iodination procedure at molecular weight (Mr) of approximately 66,000 shows uniform loading. Lanes 2 and 4 also received excess unlabeled ubiquitin. B, Quantification of high-Mr (>66,000) radioactivity (arbitrary PhosphorImager units), mean ± SEM for three independent experiments.

 
The possibility remained that insulin was itself being conjugated to ubiquitin, which would result in the production of a competitive inhibitor of lysozyme degradation. To address this question, 125I-labeled insulin was used with unlabeled ubiquitin, and unlabeled insulin with 125I-labeled ubiquitin under the same conditions, but with the addition of bacitracin, an inhibitor of insulin degradation (33) (Fig. 3Go). There was no higher molecular weight labeled material in either case, suggesting that insulin is not ubiquitin-conjugated (lanes 1–6). To be sure that bacitracin was not itself interfering with the conjugation process, 125I-ubiquitin was conjugated to lysozyme, as above, in the presence and absence of bacitracin (lanes 7–9), and no effect by bacitracin itself was seen.



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  		Figure 3. Ub is not conjugated to insulin. Ub conjugation was performed as in Fig. 2Go but with 1 mg/ml bacitracin to reduce insulin degradation. The presence or absence of ATP is indicated. A, Conjugation of ubiquitin to 125I-insulin; B, conjugation of 125I-ubiquitin to insulin; C, conjugation of 125I-ubiquitin to lysozyme without (lane 7) and with (lane 8) bacitracin. As standards, 125I-insulin (lane 3) or 125I-ubiquitin (lanes 6 and 9) alone were also applied.

 
To observe the influence of insulin in cells, we tested the effect of insulin on the ubiquitin pathway in cultured human hepatoma (HepG2) cells. Previous studies showed that treatment of cell cultures with proteasome inhibitors caused an increase in the accumulation of high-molecular-weight ubiquitin-protein conjugates, as determined by immunoblotting with ubiquitin-specific antibodies (34). Using a similar approach, we examined the accumulation of ubiquitin-protein conjugates in HepG2 cells. The levels of ubiquitin-protein conjugates in lysates from treated and untreated cells were monitored by Western blot analysis using an antiubiquitin antibody that is reactive with both free ubiquitin and ubiquitin ligated to proteins (30). Insulin, at physiologic doses, increased accumulation of high-molecular-weight ubiquitin conjugates (Fig. 4AGo). After densitometry and correction for protein loading, the data are quantified in Fig. 4BGo. Insulin treatment resulted in an 80% increase in the level of ubiquitin-protein conjugates at 1.0 nM insulin.



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  		Figure 4. Insulin increases accumulation of ubiquitin-protein conjugates in HepG2 cells. HepG2 cells were treated with the indicated amounts of insulin (Ins) for 2 h, then lysates were prepared and analyzed by Western blotting with C59 ubiquitin-specific antibody and a tubulin-specific antibody, as described in Materials and Methods. A, Ub immunoreactive proteins were detected by enhanced chemiluminescence. As a loading control, the same blot was reprobed with an antitubulin monoclonal antibody (bottom panel). B, High Mr (>116,000) was quantified by densitometry and corrected for protein loading. Data are the arbitrary densitometer units, means ± SEM (n = 4).

 
Previous studies, using purification schemes designed for the isolation of IDE, revealed copurification of the proteasome with IDE from skeletal muscle extracts (16, 17). Because insulin inhibited the ubiquitin pathway, we sought to examine the relationship between IDE and the 26S proteasome. Using a purification series designed for the enrichment of the 26S proteasome (31), proteasome activity, measured with the fluorogenic peptide substrate LLVY, copurified with insulin-degrading activity (Table 1Go). Similarly, IDE immunoreactive material (Fig. 5Go, upper panel) increased with each level of purification, in parallel with the level of proteasome-immunoreactive material (lower panel). In the final 100,000 x g centrifugation step, most of the LLVY-degrading activity and proteasome immunoreactivity was present in the pellet, whereas insulin-degrading activity and IDE immunoreactive material distributed to both the supernatant and pellet. The distribution of IDE in the pellet (19.5% of the total activity, 53.3% of the total immunoreactivity) was much higher than that expected from simple contamination of the pellet (4.0% of the total volume). These data suggest the existence of two subpopulations, both free IDE and proteasome-associated IDE, in these preparations.


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  		Table 1. Copurification of IDE and proteasome activities in a skeletal muscle 26S proteasome purification scheme

 


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  		Figure 5. Copurification of IDE with the 26S proteasome. Rat skeletal muscle extract was purified as described in Materials and Methods. Equal amounts of protein were applied to 7.5% (upper panel) or 12.0% (lower panel) SDS-PAGE and analyzed by Western blotting for the presence of IDE or proteasome, using the 9B12 monoclonal anti-IDE antibody or anti-20S proteasome polyclonal antiserum, as described in Materials and Methods.

 
To explore this further, both the supernatant and pellet from the 100,000 x g spin were applied to 10–40% glycerol gradients. With the 100,000 x g supernatant, the SDS-activated LLVY-degrading activity sediments, as a single peak in the 20S region (Fig. 6AGo), corresponding to proteasome-immunoreactive material (Fig. 6BGo). Insulin-degrading activity and IDE immunoreactivity sediment in a separate peak. In the 100,000 x g pellet, LLVY-degrading activity (Fig. 7AGo) was present in both an SDS-activated peak, consistent with the 20S proteasome (as in Fig. 6AGo), and shoulder of SDS-insensitive activity, consistent with the 26S proteasome. Proteasome immunoreactivity was present in both areas. Insulin-degrading activity and IDE immunoreactivity were present in two distinct peaks, one of which cosedimented with the 20S proteasome region. A small amount of insulin-degrading activity was present in the 26S region, and IDE immunoreactive material was detected in the 26S region, on overexposure of the blots (not shown). The other peak of insulin-degrading activity and IDE immunoreactivity sedimented in the same region of the gradient as that of the 100,000 x g supernatant material (compare with Fig. 6Go). Therefore, the data suggest that IDE, in the 100,000 x g pellet, consists of both proteasome-associated and free IDE, whereas the IDE in the 100,000 x g supernatant consists of a single species not associated with the proteasome.



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  		Figure 6. Glycerol gradient analysis of 100,000 x g supernatant. The 100,000 x g supernatant step of the 26S purification series was applied to a 10–40% glycerol gradient. A, Fractions were assayed for insulin-degrading activity (•) or LLVY-degrading activity in the presence of 0.02% SDS ({square}). There was no detectable LLVY-degrading activity without SDS. The regions of sedimentation of 26S and 20S proteasome are indicated. B, The fractions were analyzed by 7.5% (upper panel) or 12.0% (lower panel) SDS-PAGE and Western blotting to detect the presence of IDE (upper panel) or proteasome (lower panel) using the 9B12 monoclonal anti-IDE antibody or anti-20S proteasome polyclonal antiserum, as described in Materials and Methods.

 


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  		Figure 7. Glycerol gradient analysis of 100,000 x g pellet. The 100,000 x g pellet step of the 26S purification series was applied to a 10–40% glycerol gradient. A, Fractions were assayed for insulin-degrading activity (•) or LLVY-degrading activity in the absence ({blacksquare}) or presence ({square}) of 0.02% SDS. Regions of sedimentation of 26S and 20S proteasome are indicated. B, The fractions were analyzed by 7.5% (upper panel) or 12.0% (lower panel) SDS-PAGE and Western blotting to detect the presence of IDE (upper panel) or proteasome (lower panel) using the 9B12 monoclonal anti-IDE antibody or anti-20S proteasome polyclonal antiserum, as described in Materials and Methods.

 
The demonstration of IDE associated with the proteasome supports our previous reports suggesting that IDE mediates inhibition of the proteasome by insulin (19, 20, 21). A possible mechanism for this insulin action might be alteration of IDE association with the proteasome. If this were the case, then insulin treatment of the IDE-proteasome complex (100,000 x g pellet) would displace IDE from the proteasome and alter its sedimentation pattern. To test this hypothesis, the 100,000 x g pellet was incubated with and without 1 nM insulin, then analyzed by glycerol gradients (Fig. 8Go). Without insulin, insulin-degrading activity sedimented with the same profile as in Fig. 7Go, but 1 nM insulin caused IDE to sediment almost completely as the free form. Insulin has no effect on the sedimentation of SDS-sensitive proteasome activity (dotted line). This shift in the sedimentation profile of IDE is reflected in Western blots of IDE. This finding suggests that insulin treatment displaces IDE from the proteasome and produces predominantly a single species, of IDE, not associated with the proteasome.



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  		Figure 8. Displacement of IDE from the proteasome by insulin treatment. Glycerol density gradients of 100,000 x g pellet were treated without and with 1 nM insulin for 2 h at 37 C. A, The level of insulin-degrading activity was measured in the absence ({circ}) or presence (•) of insulin. The level of SDS-activated proteasome activity is indicated by the dotted line. B, Fractions from the glycerol gradients were probed for IDE and proteasome by Western blotting using the 9B12 monoclonal anti-IDE antibody or anti-20S proteasome polyclonal antiserum, as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have expanded our previous findings of insulin regulation of proteasome activity (17, 18) to the ubiquitin-proteasome pathway, a system involved in a number of critical cellular processes, including transcription factor regulation, antigen presentation, and cell cycle control (see Ref. 8 for review). Insulin inhibited ATP- and ubiquitin-dependent degradation in an in vitro extract, at concentrations similar to those seen previously for peptide degradation by the proteasome (17). Furthermore, the inhibition correlated with the level of insulin degradation in the system and was eliminated in the presence of an anti-IDE antibody, suggesting the involvement of IDE. Although the IC50 for inhibition by insulin (50 nM) was somewhat high, relative to circulating levels of insulin (10 pM to 1 nM), the lysozyme degradation assay is slow, and it requires enzyme levels above those for optimal enzyme-substrate kinetics (22). On the other hand, the cell culture experiments (Fig. 4Go) showed insulin inhibition of the ubiquitin pathway well within the physiological range of insulin. Because the ubiquitin-proteasome pathway requires conjugation of ubiquitin to substrates, insulin could have been inhibiting the ubiquitin-conjugation step, but our results showed that insulin, at doses sufficient to inhibit proteolysis, did not reduce ubiquitin conjugation to the substrate protein (Fig. 2Go), and there was no evidence of the formation of competitive ubiquitin-insulin conjugates (Fig. 3Go). This leaves the degradation step as the site of action by insulin. In cultured cells, physiological levels of insulin induced the accumulation of ubiquitin-protein conjugates (Fig. 4Go), consistent with inhibition of the ubiquitin-dependent degradation pathway.

There are several possible mechanisms for insulin regulation of proteasome activity. Insulin has been implicated in the control of levels of mRNA for ubiquitin, proteasome, and ubiquitin-conjugating enzyme (35, 36, 37), but this has been suggested to be via activation of the insulin-like growth factor (IGF)-I receptor (38). Though these mechanisms may indeed contribute to insulin regulation of proteolysis, they do not explain how insulin inhibits the ubiquitin-proteasome pathway using in vitro cell extracts, nor do they explain rapid regulation of cellular proteolysis by insulin. Another possibility is phosphorylation of the proteasome, in response to insulin, although the effect of phosphorylation on proteasome catalytic activity is conflicting (39, 40, 41). Again, this mechanism is not supported by the earlier in vitro studies, because phosphorylation should not occur under these conditions.

A novel possibility is that insulin may inhibit the proteasome through an associated protein, IDE. The catalytic properties of the proteasome can vary widely, depending on its association with regulatory proteins (42). Previously, we showed insulin inhibition of the proteasome in vitro and in cultured cells (17, 18, 19, 21). Removal of IDE from the extracts or introduction of a neutralizing antibody into cells resulted in a loss of insulin regulation of the proteasome (17, 19, 21). In the present study, using a preparation designed to enrich for the 26S proteasome from muscle, we found copurification of IDE by both insulin-degrading activity (Table 2Go) and by detection with an IDE-specific antibody (Fig. 5Go). Although this purification series is designed to purify 26S proteasome, glycerol gradient analysis of the 100,000 x g pellet showed that the majority of the proteasome sedimented in the 20S region, with much less in the 26S region, a result of rapid equilibration between the two forms, as shown previously (43). In this 100,000 x g preparation, IDE was found to cosediment with the 20S proteasome on glycerol gradients (Fig. 7Go), as well as with the 26S form when the blots were overexposed. Because of the rapid dissociation of the 26S proteasome, attempts to reapply the 26S-sedimenting material to conclusively show IDE association were unsuccessful, and thus the possibility that the IDE detected in the 26S region is caused by contamination by 20S-associated IDE cannot be ruled out. Nevertheless, the copurification of IDE through several steps, including the 100,000 x g pelleting step, suggests that IDE may be associated with the 26S, as well as the 20S proteasome forms. On the other hand, glycerol gradients using the 100,000 x g supernatant, presumably free of 26S proteasome, showed only migration of free IDE in a region that did not correspond to either the 20S or 26S proteasome (Fig. 6Go). These data suggest that IDE can exist in at least two separate populations, one associated with proteasomes, and another free form, with an altered conformation that results in a different sedimentation pattern on glycerol density gradients. This finding may explain the widely disparate reports of characteristics of IDE, including molecular size and catalytic properties (24, 44).


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  		Table 2. Continuation of Table 1Go

 
If insulin inhibition is mediated by IDE, then insulin treatment might alter IDE and, as a result, its sedimentation on glycerol density gradients. Indeed, insulin treatment (Fig. 8Go) resulted in displacement of IDE from the proteasome-associated regions to the sedimentation area of free IDE. These data suggest a shift in IDE conformation after treatment with insulin, resulting in dissociation of IDE from the proteasome, and the data imply a greater activity of the proteasome when associated with IDE. This notion is supported by our previous data, which showed a reduction in proteasome activity when it was separated from IDE by chromatography, and an elevation when they were recombined (17). The displacement of IDE from the proteasome provides a means by which insulin may regulate the activity of the 26S and 20S proteasomes. Insulin treatment of the complex results in removal of a proteasome regulatory protein, IDE. Because previous studies (21) showed inhibition of non-ATP-dependent proteasome activity by other high-affinity IDE substrates (e.g. IGF-II), whereas lower-affinity substrates (e.g. IGF-I) had little or no effect, experiments are currently underway to determine whether these other IDE substrates have effects on ATP-dependent degradation and IDE-proteasome association, or if these effects are specific for insulin. Last, though the possibility remains that one or more fragments of insulin act to indirectly inhibit the proteasome, previous studies suggested that insulin fragments generated by IDE have little effect on proteasome activity (21).

Because IDE copurified with the proteasome through the 100,000 x g precipitation step and was detected in the 26S region and the 20S region on glycerol gradients, these data are the first to suggest that IDE may be associated with both the 26S and 20S proteasomes. Because the 20S catalytic core is a common element of both forms of the proteasome, it seems likely that IDE associates with this catalytic core as an accessory or regulatory protein, which would provide a mechanism by which insulin inhibits both ATP-dependent and ATP-independent proteasome activity. The presence of IDE with the proteasome is accompanied by an elevated level of activity, possibly through an allosteric effect on the proteasome. Insulin treatment results in IDE dissociation from the proteasome and a reduction in proteasome activity. This mechanism would be consistent with the effects seen with in vitro extracts and in cultured cell experiments. Furthermore, this mechanism supports earlier results suggesting that IDE was responsible for mediating insulin regulation of the proteasome (17, 19, 20, 21).

There are a number of possible reasons why other studies have not detected the presence of IDE in proteasome preparations. Many of the routine proteasome purification strategies use EDTA in the buffers. Because IDE is a metalloproteinase, EDTA treatment causes inhibition of the insulin-degrading activity of IDE. Furthermore, EDTA treatment results in a reduction in the ability of IDE to associate with the proteasome (20). In addition, the use of anion exchange as a late step in the purification procedure can itself result in dissociation of the proteasome from IDE (16, 17). Finally, as shown in the present studies, the level of insulin degradation in the 26S region of the glycerol gradients is low, compared with that in the 20S and free regions, possibly because of inhibition of IDE by ATP (45).

Although the regulation of protein turnover by insulin has long been established, the proteolytic pathways involved have been unclear. Our results provide the first demonstration that insulin inhibits ATP- and ubiquitin-dependent degradation by the proteasome in vitro and in cultured cells. These findings extend the cellular processes controlled by insulin to include the ubiquitin-proteasome pathway, a major cellular degradation system. Because of the growing awareness of the importance of the ubiquitin pathway in the control of cellular processes, these data provide new understanding into the processes involved in a major action of insulin, the control of protein levels by regulation of proteolysis.


   Footnotes
 
1 This work was funded, in part, by the Department of Veterans Affairs Research Service and, in part, by the Bly Memorial Research Fund, University of Nebraska Medical Center. Back

2 Current address: Section of Metabolism and Endocrinology, Carl T. Hayden Veterans Affairs Medical Center, 650 East Indian School Road, Phoenix, Arizona 85012. Back

Received August 27, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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