Insulin Inhibition of the Proteasome Is Dependent on Degradation of Insulin by Insulin-Degrading Enzyme

399 Insulin inhibition of the proteasome is dependent on degradation of insulin by insulin-degrading enzyme R G Bennett1,2, J Fawcett3,4, M C Kruer3, W C Duckworth3,4,5 and F G Hamel1,2 1Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA 2Department of Medical Research, Veterans Affairs Medical Center, Omaha, Nebraska, USA 3Endocrinology Section, Carl T Hayden VA Medical Center, Phoenix, Arizona, USA 4Molecular and Cellular Biology Program, Arizona State University, Tempe, Arizona, USA 5Department of Medicine, University of Arizona, Tucson, Arizona, USA (Requests for offprints should be addressed to R G Bennett; Email: [email protected]) Abstract iodine-125 (125I)-labeled insulin, but AspB10 and EQF were somewhat more effective. All agents inhibited cross- A consequence of insulin-dependent diabetes mellitus is linking of 125I-insulin to IDE, suggesting that all were the loss of lean muscle mass as a result of accelerated capable of IDE binding. In contrast, although insulin and proteolysis by the proteasome. Insulin inhibition of lispro were readily degraded by IDE, AspB10 was degraded proteasomal activity requires interaction with insulin- more slowly, and EQF degradation was undetectable. degrading enzyme (IDE), but it is unclear if proteasome Both insulin and lispro inhibited the proteasome, but inhibition is dependent merely on insulin–IDE binding or AspB10 was less effective, and EQF had little effect. In if degradation of insulin by IDE is required. To test the summary, despite effective IDE binding, EQF was poorly hypothesis that degradation by IDE is required for protea- degraded by IDE, and was ineffective at proteasome some inhibition, a panel of insulin analogues with variable inhibition. These data suggest that insulin inhibition susceptibility to degradation by IDE binding was used to of proteasome activity is dependent on degradation by assess effects on the proteasome. The analogues used were IDE. The mechanism of proteasome inhibition may be [LysB28, ProB29]-insulin (lispro), [AspB10]-insulin (AspB10) the generation of inhibitory fragments of insulin, or by and [GluB4, GlnB16, PheB17]-insulin (EQF). Lispro was displacement of IDE from the proteasome. as effective as insulin at inhibition of degradation of Journal of Endocrinology (2003) 177, 399–405 Introduction contributing mechanism may be altered expression of mRNA for proteins involved in the ubiquitin pathway Insulin-dependent diabetes mellitus (IDDM) is character- of protein degradation, such as proteasome subunits, ized by loss of lean muscle mass caused by increased ubiquitin and ubiquitin-conjugating enzymes (Wing & proteolysis (Smith et al. 1989). The protein loss occurring Banville 1994, Price et al. 1996, Lecker et al. 1999). in IDDM is reversible with insulin treatment (Rooyackers However, these changes in the various mRNA species & Nair 1997, Grizard et al. 1999). Although insulin have not been consistently observed or always correlated induces synthesis of some proteins, the increase in cellular with changes in proteasome activity (Larbaud et al. 1996, protein induced by the hormone is attributable mostly to 2001, Wing & Bedard 1996). In addition, there is evi- a decrease in the rate of overall cellular proteolysis (Gelfand dence that insulin can decrease the catalytic activity of the & Barrett 1987). The major contributor to protein loss in proteasome: it has been found to inhibit the peptide- IDDM is an increase in the activity of the proteasome degrading activity of the proteasome in cell-free extracts (Price et al. 1996, Merforth et al. 1999, Galban et al. 2001). and in intact cells (Duckworth et al. 1994, 1998a, Hamel The proteasome is a macromolecular complex that is et al. 1997, Li et al. 2000). Furthermore, this same responsible for degradation through the energy-dependent inhibition by insulin was seen when the ATP- and ubiquitin pathway (Coux et al. 1996). The increased ubiquitin-dependent activity of the 26S form of the proteasome activity is suppressible with insulin treatment, proteasome was measured both in a cell-free system and in suggesting that the proteasome pathway is the primary cell culture (Bennett et al. 2000b). target of the anti-catabolic effect of insulin (Rooyackers & The ability of insulin to inhibit proteasome activity Nair 1997, Grizard et al. 1999, Galban et al. 2001). One requires the presence of insulin-degrading enzyme (IDE). Journal of Endocrinology (2003) 177, 399–405 Online version via http://www.endocrinology.org 0022–0795/03/0177–399 2003 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 11:26:23AM via free access 400 R G BENNETT and others · Proteasome inhibition by insulin analogues Figure 1 Sequence of human insulin and the substituted amino acids in the analogues used in the study. Amino acid substitutions are shown for lispro (gray), AspB10 (white), and EQF (black). The primary and secondary IDE cleavage sites are indicated by the heavy and light arrows respectively. IDE and the proteasome co-purify through a number of Materials and Methods chromatographic steps (Duckworth et al. 1994, Bennett et al. 2000b). When IDE is either removed from the Preparation of IDE–proteasome proteasome or inhibited, insulin has no influence on Partially purified preparations of rat skeletal muscle extract proteasome activity (Duckworth et al. 1994, 1998a, Hamel containing both IDE and proteasome were prepared as et al. 1998). In addition to insulin, other IDE substrates, described previously (Bennett et al. 2000a). Briefly, male such as atrial natriuretic peptide and relaxin, also inhibit Sprague–Dawley rats (140–160 g) were killed by decapi- the proteasome (Bennett et al. 1997). A key observation in tation under pentobarbital anesthesia, the hind leg muscle this study was that proinsulin also inhibited the protea- ff was excised and homogenized, and the cytosolic fraction some, but less e ectively than insulin. It was shown was purified by 30–60% ammonium sulfate fractionation previously that, although proinsulin binds to IDE with an ffi followed by weak anion exchange chromatography. In this a nity similar to that of insulin, it is itself a poor substrate preparation, IDE is the only insulin-degrading enzyme for degradation by IDE, being degraded much more activity. For highly purified IDE preparations, the material slowly than insulin (Duckworth et al. 1972). This suggests ffi was further purified by hydrophobic interaction, that binding to IDE is insu cient to induce proteasome chromatofocusing and strong anion-exchange chromatog- inhibition, and that IDE must degrade the substrate to raphy steps, as described previously (Bennett et al. 2000a). result in proteasome inhibition. All determinations were made using independently To explore the relationship between IDE binding and prepared enzyme samples. All animals were used in degradation and the ability of insulin to inhibit proteasome accordance with the US National Institutes of Health activity, we used a panel of insulin analogues, all based on Guide for the Use and Care of Laboratory Animals, and all biosynthetic human insulin (Fig. 1). The [AspB10]-insulin B10 B4 B16 B17 procedures were approved by the Omaha VA Medical (Asp ) and [Glu , Gln , Phe ]-insulin (EQF) ana- Center Institutional Animal Care and Use Committee. logues were developed as potential ‘long-acting’ insulin replacements (Slieker et al. 1992). Both AspB10 and EQF contain amino acid substitutions at key sites of cleavage by Insulin degradation assay IDE, and thus were selected for probable resistance to degradation by IDE. The analogue [LysB28, ProB29]- The degradation of 125I-insulin was measured by the insulin (lispro) has increased solubility compared with trichloroacetic acid solubility assay (Duckworth & native insulin (DiMarchi et al. 1994). Lispro has no Kitabchi 1974), using appropriate concentrations of substitutions at IDE cleavage sites, and was included for partially purified enzyme preparation to limit insulin comparison with AspB10 and EQF. In this study, it was degradation to the linear boundaries of the assay (c25%). found that these analogues bind to IDE, but differ in their Competitive inhibition of insulin degradation was carried ability to be degraded by IDE. This approach allowed out by including unlabeled biosynthetic human insulin or distinction between the ability of insulin or its analogues to an insulin analogue. Insulin and insulin analogues, and inhibit the proteasome, and to relate this effect to their [125I-A14]-labeled insulin were provided by Drs Ronald relative degradation by IDE. Chance and Bruce Frank of Lilly Research Laboratories. Journal of Endocrinology (2003) 177, 399–405 www.endocrinology.org Downloaded from Bioscientifica.com at 09/27/2021 11:26:23AM via free access Proteasome inhibition by insulin analogues · R G BENNETT and others 401 Covalent cross-linking of 125I-insulin to IDE Preparations of partially purified IDE were covalently cross-linked to 125I-insulin using the homobifunctional amine-reactive cross-linker, disuccinimidyl suberate (Pierce, Rockford, IL, USA), by a procedure described previously (Shii et al. 1985). To determine the specificity of binding, excess (0·1 mg/ml) unlabeled insulin or an insulin analogue was included in appropriate samples. The samples were diluted in reducing SDS-PAGE sample buffer, resolved on 7·5% SDS-PAGE, and dried. Bands of radioactivity were visualized by PhosphorImager analysis. Insulin and analogue degradation analysis using Tris/Tricine

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