WO1999035169A2 - Methods and compositions for treating and diagnosing insulin related disorders - Google Patents

Abstract

The present invention relates to methods and compositions for treating or reducing the symptoms of a disorder of absolute or relative insulin deficiency, severe insulin resistance, of lipid accumulation or excess lipid synthesis, or of protein catabolism or degradation. The invention also includes methods for detecting and for assessing treatments of such disorders.

Description

METHODS AND COMPOSITIONS FOR TREATING AND DIAGNOSING INSULIN RELATED DISORDERS

Background of the Invention

Cellular protein degradation is a complex set of interacting processes. Protein catabolism has profound clinical implications. Many pathological conditions are associated with increased protein breakdown, frequently to the detriment of the patient. Excessive protein and/or muscle breakdown occurs in uncontrolled diabetes (e.g. loss of muscle mass in a diabetic subject), severe stress (e.g., trauma, burns, sepsis), acute myocardial infarction, chronic wasting diseases (e.g., AIDS, cancer), and other conditions and diseases.

While lysosomal proteolysis has classically been considered the major site for cellular protein degradation, recent studies have shown an important role for cytosolic proteolytic processes, particularly in selective proteolytic pathways, such as the degradation of targeted proteins and short-term alterations in cellular proteolysis (i.e., inhibition by insulin). Proteasomes and lysosomes are the two major subcellular proteolytic compartments. Both of these compartments are affected by insulin. Under extreme conditions (total lack of insulin) lysosomal autophagy is activated, but under most physiological and pathophysiological conditions (e.g., stress) proteasomes may be the major target of insulin (e.g., postprandial reduction of proteolysis).

The prosome is a ubiquitous cylindrical organelle found in the cytosol of essentially every cell type in every organism, making up 1-2% of the total cellular protein. When it was discovered that the prosome was identical to the high- molecular-weight multicatalytic proteinase (MCP), the name was changed to proteasome. Multicatalytic proteinase has multiple distinct catalytic sites (as many as five) and a characteristic banding pattern on SDS gels with multiple bands in the 20-35 kDa range. Multicatalytic proteinase and its components are part of a complex proteolytic system including different molecular forms (15S, 20S, and 26S) and different pathways (ATP-dependent, ubiquitin, and non-ATP-dependent). Intense interest in this system has developed and is rapidly expanding. Recent evidence supports the proteasome, a cytosolic proteolytic complex, as a central component in cellular protein turnover. Proteasome activity is important for ubiquitin-mediated proteolysis, insulin-altered proteolysis, antigen processing, apoptosis, cell growth and differentiation, and many other proteolvsis-dependent cell functions. Although multicatalytic proteinase has been implicated in almost all cellular degradative functions, relatively little is known about its control.

The insulin-degrading enzyme (IDE) insulinase was first identified by its relatively high specificity for the degradation of insulin. Subsequently, other substrates for insulin-degrading enzyme have been recognized, although insulin has the highest affinity for the enzyme. Characterization of the enzyme proved difficult, with widely varying reports of such basic properties as molecular weight, type of proteolytic activity, and pH optimum. In particular, purification of the protein was elusive because of instability and variable properties depending on the approach. The purification to homogeneity and the subsequent isolation of the cDNA has clarified some of the issues. Insulin-degrading enzyme has no homology to classical proteinases, but rather is the initial representative of a proposed new superclass of metalloproteinases with a requirement for Zn²⁺ but without the typical Zn²⁺ binding site. Insulin-degrading enzyme has a molecular weight of about 1 10,000 Daltons. The insulin-degrading enzyme is important for the cellular processing and degradation of insulin. The general characteristics of cellular insulin degradation are consistent with the properties of this enzyme, and insulin-degrading enzyme is present in endosomes where insulin degradation begins. Cellular degradation products of insulin are consistent with the known cleavage sites of insulin-degrading enzyme. However, the primary cellular location of insulin-degrading enzyme is cytosolic, and the enzyme is present in all cell types examined, including cells that do not bind and internalize insulin, suggesting that insulin-degrading enzyme has a broader function than simply insulin degradation. This concept is supported by the presence of insulin-degrading enzyme in organisms from Escherichia coli to humans and its evolutionary conservation. Insulin-degrading enzyme has also been shown to be developmental^ regulated and has been implicated in cell differentiation and growth.

It has been shown that insulin-degrading enzyme can regulate the activity of the multicatalytic proteinase or proteasome. Insulin-degrading enzyme and multicatalytic proteinase can be isolated from cytosol as a complex. Under conditions that maintain the association of these enzymes, insulin inhibits multicatalytic proteinase degradation of some but not all of the substrates of this multicatalytic enzyme. After the separation of insulin-degrading enzyme and multicatalytic proteinase by purification, the insulin effect is lost. The insulin degrading enzyme, the multicatalytic proteinase, and their complex are implicated in protein catabolism.

However, general acceptance of an intracellular action of insulin has not occurred nor has such action been investigated. The lack of a known mechanism for producing intracellular effects of insulin has limited approaches to solving these problems. Given the important role of proteasomes in mediating cellular proteolysis, a system to modify and to assess modification of proteasome activity would have important clinical implications in conditions associated with altered protein catabolism, such as diabetes, stress, AIDS, cancer, etc., and there remains a need for such systems.

Summary of the Invention The present invention relates to methods for treating or reducing the symptoms of a disorder of absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject), of severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or of protein catabolism or degradation (e.g. loss of muscle mass in a subject with diabetes or a wasting disease), and to peptides that can be employed in such a method. A preferred method of treating or reducing symptoms of such a disorder, in a patient in need thereof, includes administering to the patient a polypeptide including a sequence flanking an insulin degrading enzyme cleavage site of insulin. Such peptides preferably inhibit one or more activities of a complex of insulin degrading enzyme and multicatalytic proteinase. In one embodiment, the method of treating or reducing symptoms can include administering a peptide that inhibits an activity of a complex of insulin degrading enzyme and multicatalytic proteinase. The invention also includes methods of detecting a disorder of absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject), of severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or of protein catabolism or degradation (e.g. loss of muscle mass in a subject with diabetes or a wasting disease). Such a method of detecting includes measuring, in a biological sample derived from a patient, an activity of a complex of insulin degrading enzyme and multicatalytic proteinase. In a preferred method of detecting, the level of this activity is compared to a level for a suitable control group. In one embodiment, the method can include measuring the effects of inhibitors on an activity of a complex of insulin degrading enzyme and multicatalytic proteinase.

In another embodiment of the invention, measuring, in a biological sample derived from a patient, an activity of a complex of insulin degrading enzyme and multicatalytic proteinase can be employed to assess the effectiveness of a treatment of a disorder of absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject), severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or of protein catabolism or degradation (e.g. loss of muscle mass in a subject with diabetes or a wasting disease). In this method, the level of this activity is compared to a suitable control to determine whether the patient's level has returned to or entered a typical range.

Brief Description of the Drawings

Figure 1 illustrates that peptides that are high-affinity ligands of insulin- degrading enzyme inhibit LLV Y degradation by the complex of insulin degrading enzyme and multicatalytic proteinase. The enzyme complex (D) or purified multicatalytic proteinase (■) was incubated in the presence and absence of 1 μmol/l peptide hormones. The effect of the peptide-hormone on the degradation by the complex versus purified multicatalytic proteinase was compared (*P<0.01). LLVY degradation is expressed as a percentage of the fluorescence liberated (arbitrary units) per 60 minutes, compared with the addition of vehicle only. Data are shown as a means + SE for the three independent experiments. GLC, glucagon; INS, insulin; PRO, proinsulin; RXN, relaxin.

Figure 2 presents results showing that peptides that are high-affinity ligands of insulin-degrading enzyme inhibit LSTR degradation by the complex of insulin degrading enzyme and multicatalytic proteinase. The enzyme complex (D) or purified multicatalytic proteinase (■) were incubated in the presence and absence of 1 μmol/l peptide hormones. The effect of the peptide hormone on the degradation by the complex versus purified multicatalytic proteinase was compared (*P<0.05, **P<0.01). LSTR degradation is expressed as a percentage of the fluorescence liberated (arbitrary units) per 60 minutes compared with the addition of vehicle only. Data are shown as means + SE for the three independent experiments.

Abbreviations used include: GLC, glucagon; INS, insulin; PRO, proinsulin; RXN, relaxin.

Figure 3 shows peptides that have little effect on LLE degradation by the multicatalytic proteinase. The enzyme complex (D) or purified multicatalytic proteinase (■) were incubated in the presence and absence of 1 μmol/l peptide hormones. The effect of the peptide hormone on the degradation by the complex versus purified multicatalytic proteinase was compared (*P<0.05). LLE degradation is expressed as a percentage of the fluorescence liberated (arbitrary units) per 60 minutes, compared with addition of vehicle only. Data are shown as means + SE for the three independent experiments. Abbreviations used include: GLC, glucose; INS, insulin; PRO, proinsulin; RXN, relaxin.

Figures 4 A and 4B present dose response curves of the effect of insulin and analogs on LLVY degradation (A) and LSTR degradation (B) by the complex of insulin degrading enzyme and multicatalytic proteinase. Insulin and lys-pro insulin have approximately equivalent effects. BlO-asp inhibits less, and EQF shows no significant inhibition. Figures 5A and 5B illustrate dose response curves of the effect of insulin and peptides on IDE-MCP catalyzed by hydrolysis is of LLYY (A) and LSTR (B). HLVEALY has a triphasic effect with the greatest inhibition at 10^"5M. LVEALY inhibits at 10^"5M. Figure 6 illustrates dose response effects for inhibition of degradation of ¹²⁵I insulin catalyzed by the MCP-IDE complex. Insulin, insulin analogs, and insulin- derived peptides were tested. B 10-asp insulin and wild type insulin are equivalent. EQF insulin is more effective. The peptides HLVEALY and LVEALY inhibit at concentrations in the range of 10^"6 - 10^"5M. Figure 7 illustrates dose-dependent inhibition of FLF degradation by insulin in intact HepG2 cells. Subconfluent cultures of HepG2 cells were serum-deprived overnight, then treated with the indicated concentrations of insulin for two hours, followed by addition of substrate (FLF) for one hour. Data are expressed as mean + SEM of % FLF degradation with vehicle only, for at least four independent experiments. The EC₅₀ is 1.5 x 10^"13M insulin, with a maximal inhibition 19.1%. ** Indicates that the results are significantly different from no insulin added (pθ.01).

Figures 8 A and 8B illustrate inhibition of FLF degradation by the complex of insulin degrading enzyme and multicatalytic proteinase in HepG2 cells by insulin and insulin-derived peptides. Dose response curves of insulin, HLVEALY and LVEALY are shown at 60 (A) and 120 minutes (B) after addition of insulin or the peptide.

Figure 9 illustrates inhibition by insulin of FLF degradation by the complex of insulin degrading enzyme and multicatalytic proteinase in isolated hepatocytes over time as % of zero time in the absence of added insulin.

Figures 10A and 10B show the effects of LVEALY (A) and HLVEALY (B) on FLF degradation by the complex of insulin degrading enzyme and multicatalytic proteinase in isolated hepatocytes expressed as % of degradation without added peptide at time zero. Figure 1 1 illustrates inhibition of total cell protein degradation in H4 cells by insulin, an insulin analog, and an insulin-derived peptide. Cells were labeled overnight and then washed; then the effect of insulin, analogs, and peptides was measured.

Figure 12 illustrates inhibition of total cell protein degradation in H4 cells by insulin, insulin analogs, and an insulin-derived peptide after a three-hour preincubation in the absence of label. Cells were labeled overnight and washed. After a three-hour preincubation protein degradation over four hours was assessed.

Figure 13 illustrates urine output by control and HLVEALY-treated animals.

Figure 14 illustrates body weight of control and HLVEALY-treated animals.

Figure 15 illustrates urine output by control and HLVEALY-treated animals. Figure 16 illustrates body weight of control and HLVEALY-treated animals.

Figure 17 illustrates the initial blood glucose levels in diabetic animals treated with insulin or insulin-derived peptide.

Figure 18 illustrates the final blood glucose levels in diabetic animals treated with insulin or insulin-derived peptide. Figure 19 illustrates urinary N-methylhistidine excretion from diabetic animals treated with insulin or insulin-derived peptide.

Figure 20 illustrates serum triglyceride levels in diabetic animals treated with insulin or insulin-derived peptide.

Figure 21 illustrates serum β-hydroxybutyrate levels in diabetic animals treated with insulin or insulin-derived peptide.

Figure 22 illustrates serum non-esterified fatty acid levels in diabetic animals treated with insulin or insulin-derived peptide.

Figure 23 shows absolute epitrocharis muscle weight in control animals and rats treated with insulin, HLVEALY, and LVEALY. Figure 24 illustrates the effect on epitrocharis muscle weight in control and experimental animals expressed as % of total body weight.

Figure 25 shows absolute epididymal fat pad weight in control animals and rats treated with insulin, HLVEALY, and LVEALY.

Figure 26 illustrates the effect on epididymal fat pad weight in control and experimental animals expressed as % of total body weight. Figure 27 illustrates the effect on epididymal fat pad weight in control and experimental animals expressed as % of total leg weight.

Figure 28 illustrates the effect on epididymal fat pad weight in control and experimental animals expressed as % of solus weight. Figure 29 illustrates the effect on epididymal fat pad weight in control and experimental animals expressed as % of epitrocharis weight.

Figure 30 illustrates changes in body weight in control animals and rats treated with insulin, HLVEALY, and LVEALY.

Figure 31 shows a dose dependent effect of insulin on decreasing protein degradation in intact cells labeled under standard conditions (18 hours labeling, 3 hours wash, and 4 hour incubation).

Figure 32 shows insulin and peptide effects on decreasing protein degradation in intact cells labeled under different conditions of labeling and treatment (no wash, incubation medium without Ca⁺⁺ but with amino acids) than those employed for Figure 31.

Figure 33 illustrates glucose incorporation into lipid in intact adipocytes treated with insulin and HLVEALY.

Figure 34 illustrates glucose oxidation in intact adipocytes treated with insulin and HLVEALY. Figure 35 illustrates the interaction among dexamethasone, insulin, and TNF and their effects on IL8 secretion from respiratory endothelial cells.

Figure 36 illustrates the effect of insulin and insulin-derived peptides on DNA sysnthesis in H4 hepatocyte cells.

Figure 37 illustrates the effect of HLVEALY treatment on urinary 3- methylhistidine excretion by obese, type 2 diabetic rats.

Figure 38 illustrates the effect of HLVEALY treatment on total fatty acid oxidation in adipocytes of obese, type 2 diabetic rats.

Figure 39 illustrates the effect of HLVEALY treatment on peroxisomal fatty acid oxidation in adipocytes of obese, type 2 diabetic rats. Detailed Description of the Invention

Insulin Degrading Enzyme

Insulin degrading enzyme is a metalloenzyme containing Zn⁺⁺, and possibly Mn⁺⁺, and its degradative activity requires one or more divalent cations. In addition to Zn⁺⁺ and Mn⁺⁺, Ca⁺⁺ has also been shown to affect the degradative activity of insulin-degrading enzyme in vitro and in intact cells. The insulin degrading enzyme cleaves insulin at sites including in the B chain between residues 9 and 10, between residues 10 and 11 , between residues 16 and 17, between residues 24 and 25, and between residues 25 and 26. While insulin is the substrate with the greatest affinity, insulin-degrading enzyme also interacts with other peptides and proteins. In general, substrates recognized by the enzyme have some structural homology with insulin (proinsulin, proinsulin intermediates, epidermal growth factor [EGF], IGF-I, IGF-II, relaxin, and atrial naturetic peptide [ANP]). This finding has led to the conclusion that the enzyme recognizes structural features of these proteins.

The insulin degrading enzyme can be purified, isolated, or studied in intact cells or organisms. The enzyme can exist as the insulin degrading enzyme, as part of a complex with the multicatalytic proteinase, or as a component of other intracellular systems. The activity of insulin degrading enzyme can be measured using a variety of assays known to those of skill in the art. A typical assay with a radiolabeled protein substrate employs trichloroacetic acid to precipitate substrate, while products remain soluble. HPLC based assays for activity of insulin degrading enzyme are also known in the art. Known assays can be used to monitor activity of insulin degrading enzyme either in vitro or in intact cells or organisms.

Multicatalytic Proteinase

Multicatalytic proteinase (MCP), also known as the proteasome, has multiple catalytic sites, including chymotrypsin-like, trypsin-like, and peptidyl-glutamyl- degrading activities, among others. These catalytic sites degrade a variety of substrates including proteins and peptides useful for in vitro and in vivo assays. The multicatalytic proteinase can be purified, isolated, or studied in intact cells or organisms. The various activities of the multicatalytic proteinase can be measured using a variety of assays known to those of skill in the art. A typical assay with a radiolabeled protein substrate employs trichloroacetic acid to precipitate substrate, while products remain soluble. There are also peptide based assays employing release of a chromogenic or fluorogenic compound from the peptide upon cleavage by multicatalytic proteinase or by a complex of multicatalytic proteinase with insulin degrading enzyme. Known assays can be used to monitor activity of multicatalytic proteinase, or the complex, either in vitro or in intact cells or organisms.

Insulin added to isolated proteasomes noncompetitively inhibits the chymotrypsin-like and trypsin-like activities. This inhibition can be employed in investigation of the effect of proteins on the activities of the multicatalytic proteinase, either alone or as part of a complex with the insulin degrading enzyme. There is an in vitro system in which the effects of modifiers of proteasome activity can be assessed directly. Insulin and related proteins have a direct effect on this system. While this has important basic research implications, of more general and clinical importance is that the system can be used to screen unrelated materials for an effect on proteolysis. For example, proteasome activity in intact cells can be assayed using a membrane permeable substrate. As a clinical assay, patient sera or other tissue can be screened for effects on proteasome activity and, importantly, the effects of treatment on proteasome activity can be assessed. Treatments which result in reduction of proteasome activity have a net beneficial effort on protein catabolism (e.g. preserving muscle weight or decreasing muscle breakdown in a subject with diabetes or a wasting disease). Similarly, decreases in proteasome activity occur in some pathological conditions, and a method for assessing effective approaches to increase activity has clinical importance.

Methods of Detecting Disorders and Assessing Treatments

Measuring levels of one or more of the activities of the complex of insulin degrading enzyme and multicatalytic proteinase can be employed in methods for detecting a disorder of absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject), severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or of protein catabolism or degradation (e.g. loss of muscle mass in a subject with diabetes or a wasting disease), or for assessing the efficacy of a treatment for such a disorder (e.g. preserving muscle weight or decreasing muscle breakdown in a subject with diabetes or a wasting disease). Methods for measuring the several activities of the complex of insulin degrading enzyme and multicatalytic proteinase are described herein and are known to those of skill in the art.

Such an activity can be measured in a suitable biological sample for detecting or assessing treatment of a disorder of absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject), severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or of protein catabolism or degradation (e.g. loss of muscle mass in a subject with diabetes or a wasting disease). Suitable biological samples include blood, plasma, pancreas, muscle, fat, liver, urine and the like. The measured level can then be compared to control levels of activity. Suitable controls include the same patient at a different time, an historical population of patients having the disorder, a predicted level, and the like. The comparison determines factors such as whether the patient suffers from the disorder, and to what degree the disorder has progressed or been successfully treated. Such assays can also be used to assess the effect on protein catabolism of actual or candidate therapeutic agents.

Insulin

The term "insulin" as used herein refers to mammalian insulin, such as bovine, porcine or human insulin, whose sequences and structures are known in the art. Bovine, porcine, and human insulin are preferred mammalian insulins; human insulin is more preferred. The amino acid sequence and spatial structure of human insulin are well-known. Human insulin is comprised of a twenty-one amino acid A- chain and a thirty amino acid B-chain which are cross-linked by disulfide bonds. A properly cross-linked human insulin contains three disulfide bridges: one between position 7 of the A-chain and position 7 of the B-chain, a second between position 20 of the A-chain and position 19 of the B-chain, and a third between positions 6 and 1 1 of the A-chain.

The term "insulin analog" means proteins that have an A-chain and a B-chain that have substantially the same amino acid sequences as the A-chain and B-chain of human insulin, respectively, but differ from the A-chain and B-chain of human insulin by having one or more amino acid deletions, one or more amino acid replacements, one or more amino acid additions, and/or one or more side chain alterations that do not destroy the insulin activity of the insulin analog.

One type of insulin analog, "monomeric insulin analog," is well known in the art. Such analogs of human insulin include, for example, human insulin in which Pro at position B28 is substituted with Asp, Lys, Leu, Vai, or Ala, and wherein Lys at position B29 is Lys or is substituted with Pro, and also, AlaB26-human insulin, des(B28-B30) human insulin, and des(B27) human insulin. Monomeric insulin analogs are disclosed in Chance, et al, U.S. Patent No. 5,514,646, issued May 7, 1996; Brems, et al, Protein Engineering, 6:527-533 (1992); Brange, et al, EPO Publication No. 214,826 (published March 18, 1987); and Brange, et al, Current Opinion in Structural Biology, 1 :934-940 (1991). These disclosures are expressly incorporated herein by reference for describing monomeric insulin analogs. The monomeric insulin analogs employed in the present formulations are properly cross- linked at the same positions as is human insulin.

Insulin analogs may also have replacements of the amidated amino acids with acidic forms. For example, Asn may be replaced with Asp or Glu. Likewise, Gin may be replaced with Asp or Glu. In particular, AsnA18, AsnA21, or AspB3, or any combination of those residues, may be replaced by Asp or Glu. Also, GlnA15 or GlnB4, or both, may be replaced by either Asp or Glu. Alternative insulin analogs are those having, optionally, among other replacements or deletions, Asp at B21, or Asp at B 3, or both replacements.

Insulin Derived Inhibitors Administration of insulin derived inhibitors of one or more activities of the complex of IDE and MCP in a condition or disorder of absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject), severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or of protein catabolism or degradation (e.g. loss of muscle mass in a subject with diabetes or a wasting disease) represent an effective method to reduce symptoms or effects of the condition or disorder. Such conditions include (but are not limited to) diabetes, severe stress (trauma, burns, starvation), myocardial infarction, and chronic wasting diseases (AIDS, cancer, etc.) Such a peptide would not be expected to have direct effects on glucose metabolism or cell growth and mitogenesis since the effects of insulin on these processes are through different mechanisms. Indirect, potentially beneficial, effects (glucose lowering, decreased mitogenesis) are possible.

Insulin inhibits certain activities of the complex of insulin degrading enzyme and the multicatalytic proteinase. Polypeptides derived from insulin also inhibit these activities of the complex. For example, polypeptide products of insulin cleavage by the insulin degrading enzyme inhibit the complex. More fully degraded insulin, for example, insulin degradation products including primarily substances soluble in trichloroacetic acid, is less effective at inhibiting the complex. This indicates that insulin derived polypeptides including an amino acid sequence flanking a cleavage site for the insulin degrading enzyme are effective inhibitors of the complex. As used herein, flanking the cleavage site refers to a polypeptide having a sequence adjacent to the cleavage site and including one of the amino acids which undergoes bond cleavage by the enzyme.

Inhibitor polypeptides including an amino acid sequence flanking a cleavage site can be as small as about 4 to about 15 amino acids, preferably about 5 to about 8 amino acids. Such preferred polypeptides include HLVEALY (SEQ ID NO: l) and LVEALY (SEQ ID NO: 2). These preferred polypeptides represent amino acids 10- 16 and 11-16, respectively, from the insulin B-chain. The insulin degrading enzyme cleaves insulin at sites including in the B chain between residues 9 and 10, between residues 10 and 11, between residues 16 and 17, between residues 24 and 25, and between residues 25 and 26. Thus, the peptide HLVEALY and LVEALY each flank two cleavage sites. Additional peptides that flank these cleavage sites include, for example, peptides representing residues 1-9, 5-9, 1-10, 7-10, 9-24, 9-25. 10-24, 10- 25. 16-21, 16-25, 16-26, 17-24, 17-25, 17-30, 24-30, and 25-30 of the insulin B chain, and the like. Certain derivatives of these peptides, such as gamma-glutamyl residues can also be included. Polypeptides, proteins and peptides of the invention can be produced by synthetic or recombinant technologies. Polypeptides smaller than about 15-20 amino acids can be conveniently made by well known methods of peptide synthesis, such as automated solid phase peptide synthesis. Such polypeptides can also be made by recombinant methods. For example, such a polypeptide can be made as part of a fusion protein, or repeats of the desired sequence can be expressed as multiple units in a long chain polymer. Polypeptides longer than about 20-90 amino acids can be conveniently produced by numerous known recombinant methods.

Suitable inhibitors of the complex of insulin degrading enzyme and multicatalytic proteinase include the polypeptides corresponding substantially to the amino acid sequences within insulin that are described above. For the purposes of the invention, the definition of the polypeptides corresponding substantially to an amino acid sequence within insulin includes peptides which correspond to an amino acid sequence within an allelic variant or mutant of insulin. The variants and mutants possess a high degree of sequence homology with the native sequence (e.g., substitution, deletion or addition mutants). Polypeptides which correspond substantially to the amino acid sequence of insulin typically have at least about 70% and more preferably at least about 90%) sequence homology with the native sequence. Preferably, polypeptides which correspond substantially to the amino acid sequence of the insulin typically have at least about 70% and more preferably at least about 90% sequence identity with the native sequence.

In addition to corresponding substantially to an amino acid sequence within insulin, the present polypeptides retain desirable properties of insulin or of inhibitory fragments of insulin. Preferably, the polypeptides of the invention maintain the functional activity of insulin bind to and/or inhibit the complex of insulin degrading enzyme and multicatalytic proteinase. In addition, the polypeptides of the invention can be products of or substrates for the insulin degrading enzyme and/or the complex of insulin degrading enzyme and multicatalytic proteinase. Analogs or derivatives of these peptides that have additional desirable characteristics (e.g., resistant to degradation or increased membrane permeability) are included in the invention as well. Compounds potentially useful can be screened employing the in vitro and other assays systems described herein.

Preferably, the present variants of insulin-derived polypeptides are modified through deletions or conservative amino acid substitutions. Typically, such conservative amino acid substitutions include substitutions such as described by Dayhoff in the "Atlas of Protein Sequence and Structure," 5, (1978) and Argos in EMBO J., 8, 779 ( 1989), the disclosures of which are herein incorporated by reference. For example, the exchange of amino acids within one of the following classes represent conservative substitutions: Class I: Ala, Gly, Ser, Thr, and Pro (representing small aliphatic side chains and hydroxyl group side chains); Class II: Cys, Ser, Thr and Tyr (side chains including an -OH or -SH group); Class III: Glu, Asp, Asn and Gin (representing carboxyl group containing side chains): Class IV: His, Arg and Lys (representing basic side chains); Class V: He, Vai, Leu, Phe and Met (representing hydrophobic side chains); Class VI: Phe, Trp, Tyr and His (representing aromatic side chains); and Class VII: Lys, Asp, Glu, Asn and Gin. The classes also include related amino acids such as 3Hyp and 4Hyp in Class I; homocysteine in Class II; 2-aminoadipic acid, 2-aminopimelic acid, g- carboxyglutamic acid, b-carboxyaspartic acid, and the corresponding amino acid amides in Class III; ornithine, homoarginine, N-methyl lysine, dimethyl lysine, trimethyl lysine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, homoarginine, sarcosine and hydroxylysine in Class IV; substituted phenylalanines, norleucine, norvaline, 2-aminooctanoic acid, 2-aminoheptanoic acid, statine and b-valine in

Class V; and naphthylalanines, substituted phenylalanines, tetrahydroisoquinoline-3- carboxylic acid, and halogenated tyrosines in Class VI.

Larger compilations of related amino acids and amino acid derivatives may be found in a variety of publications known to those skilled in the art, e.g., the catalogue of Bachem Biosciences, Inc. (King of Prussia, PA). Moreover, the classes may include both L and D stereoisomers, although L-amino acids are typically preferred for substitutions. As used herein, the term "conservative amino acid substitutions" also includes a number of other amino acid substitutions identified as frequently occurring conservative amino acid substitutions by Gribskov et al., Nucl. Acid Res., 14(16), 6745 (1986), the disclosure of which is herein incorporated by reference. Included among such conservative amino acid substitutions are the exchange of Ala with Cys, Asp or Glu; the exchange of Gly or His with Asp, Glu or Gin; the exchange of Ser with Asn, Phe or Trp; the exchange of Leu with Tyr or Trp; and the exchange of Pro with Glu, Gin or Arg. The amino acid derivative can also be a phosphorylated amino acid, a gamma-glutamyl amino acid, or another naturally occurring derivative of an amino acid. Preferably, the amino acid derivative is one naturally occurring in insulin or insulin degradation products.

Additional Polypeptide Inhibitors

Certain other proteins also inhibit one or more activities of the complex of insulin degrading enzyme and the multicatalytic proteinase. These proteins include atrial naturetic peptide, relaxin, TGFα, or insulin-like growth factor II, and certain polypeptides derived from these proteins. For example, polypeptide products of cleavage of atrial naturetic peptide, relaxin, or insulin-like growth factor II by the insulin degrading enzyme inhibit the complex. This indicates that polypeptides derived from these proteins and including an amino acid sequence flanking a cleavage site for the insulin degrading enzyme are effective inhibitors of the complex. Inhibitor polypeptides including an amino acid sequence flanking a cleavage site can be as small as about 4 to about 15 amino acids, preferably about 5 to about 8 amino acids. Suitable inhibitors of the complex of insulin degrading enzyme and multicatalytic proteinase include polypeptides with sequences corresponding substantially to the amino acid sequences for the proteins described above and derivatives of the proteins described above. Polypeptides corresponding substantially to atrial naturetic peptide, relaxin, TGFα, or insulin-like growth factor II, and certain polypeptides derived from these proteins have characteristics analogous to those described above for sequences corresponding to insulin. Pharmaceutical Compositions of Polypeptides

The present insulin-derived, and other, polypeptides can be used in pharmaceutical compositions for treatment of or reducing symptoms of disorders such as diabetes, severe stress (trauma, burns, starvation), myocardial infarction, and chronic wasting diseases (AIDS, cancer, etc.), other disorders including absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject) or severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), and/or disruption of protein degradation or catabolism (e.g. loss of muscle mass in a subject with diabetes or a wasting disease).

The pharmaceutical compositions of the present invention include an insulin- derived polypeptides in effective unit dosage form and a pharmaceutically acceptable carrier. As used herein, the term "effective unit dosage" or "effective unit dose" is denoted to mean a predetermined amount sufficient to be effective for treatment of or reducing symptoms of disorders including absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject) or severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or disorders including disruption of protein degradation or catabolism (e.g. loss of muscle mass in a subject with diabetes or a wasting disease). Pharmaceutically acceptable carriers are materials useful for the purpose of administering the medicament, which are preferably non-toxic, and can be solid, liquid, or gaseous materials, which are otherwise inert and medically acceptable and are compatible with the active ingredients.

Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic) for injectable solutions. The carrier can be selected from various oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions can be subjected to conventional pharmaceutical expedients, such as sterilization, and can contain conventional pharmaceutical additives, such as preservatives, stabilizing agents, wetting, or emulsifying agents, salts for adjusting osmotic pressure, buffers, and the like. Suitable pharmaceutical carriers and their formulations are described in Martin, "Remington's Pharmaceutical Sciences," 15th Ed.; Mack Publishing Co., Easton (1975); see, e.g., pp. 1405-1412 and pp. 1461-1487. Such compositions will, in general, contain an effective amount of the active compound together with a suitable amount of carrier so as to prepare the proper dosage form for proper administration to the host.

These pharmaceutical compositions can be administered parenterally, including by injection; orally; as a patch, with or without iontophoresis; used as a suppository or pessary; applied topically as an ointment, cream, aerosol, powder; or given as eye or nose drops, etc., depending on whether the preparation is used to treat internal or external disorders.

The compositions can contain 0.1% - 99% of the active material. For topical administration, for example, the composition will generally contain from 0.01% to 20%, and more preferably 0.5%) to 5% of the active material.

The present invention is also drawn to methods for of or reducing symptoms of disorders such as diabetes, severe stress (trauma, burns, starvation), myocardial infarction, and chronic wasting diseases (AIDS, cancer, etc.), other disorders including absolute or relative insulin deficiency (e.g. type 2 diabetes in an obese subject) or severe insulin resistance, of lipid accumulation or excess lipid synthesis (e.g. increased body fat or lipid synthesis in an obese and/or diabetic subject), or other disorders including disruption of protein degradation or catabolism (e.g. loss of muscle mass in a subject with diabetes or a wasting disease). Typically, the compositions will be administered to a patient (human or other animal, including mammals such as, but not limited to, cats, horses, pigs, sheep, dogs, and cattle and avian species) in need thereof, in an effective amount to treat or reduce the symptoms of the disorders. The present compositions can be given either orally, intravenously, intramuscularly or topically. For oral administration, fine powders or granules can contain diluting, dispersing and/or surface active agents, and can be presented in a draught, in water or in a syrup; in capsules or sacnets in the dry state or in a non-aqueous solution or suspension, wherein suspending agents can be included; in tablets or enteric coated pills, wherein binders and lubricants can be included; or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents can be included. Tablets and granules are preferred, and these can be coated.

For buccal administration, the compositions can take the form of tablets or lozenges formulated in a conventional manner.

For parenteral administration or for administration as drops, as for eye infections, the compounds can be presented in aqueous solution in a concentration of from about 0.1 to 10%, more preferably 0.5 to 2.0%, most preferably 1.2% w/v. The solution can contain antioxidants, buffers, etc.

The compositions according to the invention can also be formulated for injection and can be presented in unit dose form in ampoules or in multi-dose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free buffer saline, before use. The present compositions can also be in the form of encapsulated liposomes.

The compositions can be applied to the body of the patient as a topical ointment or cream. The compounds can be presented in an ointment, for instance with a water-soluble ointment base, or in a cream, for instance with an oil in water cream base, in a concentration of from about 0.1 to 10%, preferably 0.5 to 2.0%o, most preferably 1.2% w/v. For topical administration, the daily dosage as employed for adult human treatment will range from 0.1 mg to 1000 mg, preferably 0.5 mg to 10 mg. However, it will be appreciated that serious disorders can require the use of higher doses.

The compositions can also be applied into body orifices such as the nose, oral cavity and ears in the form of a spray or drops. For example, the compositions can be applied into body orifices such as the rectum and vagina in the form of a suppository or cream.

For systemic administration, the daily dosage as employed for adult human treatment will range from 5 mg to 5000 mg of active ingredient, preferably 50 mg to 2000 mg, which can be administered in 1 to 5 daily doses, for example, depending on the route of administration and the condition of the patient. When the compositions include dosage units, each unit will preferably contain 2 mg to 2000 mg of active ingredient, for example 50 mg to 500 mg. For serious infections, the compound can be administered by intravenous infusion using, for example, 0.01 to 10 mg/kg/hr of the active ingredient.

The present invention also encompasses a kit including the present pharmaceutical compositions and to be used with the methods of the present invention. The kit can contain a vial which contains an insulin-derived polypeptide of the present invention and suitable carriers, either dried or liquid form. The kit further includes instructions in the form of a label on the vial and/or in the form of an insert included in a box in which the vial is packaged, for the use and administration of the compounds. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow a worker in the field to administer the drug. It is anticipated that a worker in the field encompasses any doctor, nurse, or technician who might administer the drug.

The present invention also relates to a pharmaceutical composition including an insulin-derived polypeptide and suitable for administration for the purposes or uses described herein. According to the invention, an insulin-derived polypeptide can be used for manufacturing a composition or medicament suitable for parenteral or oral administration. The invention also relates to methods for manufacturing compositions including an insulin-derived polypeptide in a form that is suitable for parenteral or oral administration. For example, parenteral or oral formulation can be manufactured in several ways, using conventional techniques. A liquid formulation can be manufactured by dissolving an insulin-derived polypeptide in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients. The present invention may be better understood with reference to the following examples. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

Examples Example 1 — Insulin and Other IDE Substrates Inhibit Activities of the Complex of Insulin Degrading Enzyme and Multicatalytic Proteinase Insulin-degrading enzyme interacts with, binds to, and cleaves insulin and additional peptides and proteins. In general, substrates recognized by the enzyme have some structural homology with insulin. Examples of such substrates include proinsulin, proinsulin intermediates, epidermal growth factor (EGF), IGF-I, IGF-II, relaxin, and atrial naturetic peptide (ANP). Among these substrates, several, including proinsulin, EGF, and IGF-I, bind but are cleaved only slowly, and others, including IGF-II and ANP are readily degraded. It was of interest to determine which of these substrate polypeptides inhibit activities of the complex of insulin degrading enzyme and the multicatalytic proteinase.

Research Design and Methods [^l25I]iodoinsulin, specifically labeled on Tyr*¹⁴ or Tyr^B26, was provided by Dr.

Bruce Frank of the Eli Lilly Research Laboratory or was made by methods known in the art. Crystalline porcine insulin, glucagon, human proinsulin, IGF-I, and IGF-II were provided by Dr. Ronald Chance of the Eli Lilly Research Laboratory. Enzyme- grade ammonium sulfate was purchased from ICN Biomedicals (Irvine, CA). Fluorogenic peptides like succinyl-leu-leu-val-tyr-7-amido-4-methylcoumarin

[LLVY], CBZ-leu-leu-glu β-napthyl-amide [LLE], and boc-leu-ser-thr-arg-7 amido- 4-methylcoumarin [LSTR] were purchased from Sigma. DEAE-Sephacel, phenyl- Sepharose, Sephadex G-50, and Mono-Q were purchased from Pharmacia. Bio-Gel P-200 was purchased from Bio-Rad. All other chemicals were reagent grade or better. Preparation of enzyme samples. The complex of insulin degrading enzyme and multicatalytic proteinase was prepared from rat muscle and purified by ultracentrifugation and ammonium sulfate precipitation by methods known in the art. For some experiments, the complex was further purified with DEAE-Sephacel. Purified multicatalytic proteinase was prepared as above, followed by chromatography on phenyl-Sepharose, Bio-Gel P-200, and Mono-Q by methods known in the art.

Measurement of degradative activity. Insulin degradation using Al 4 [^,25I]iodo-insulin, or other labeled insulin, was measured by trichloroacetic acid precipitation and expressed as percent soluble per 15 minutes incubation at 37°C. The degradation of fluorogenic peptides was carried out by methods known in the art and measured by fluorescence at excitation and emission wavelengths of 390 and 440 nm (LLVY and LSTR) or 335 and 410 ran (LLE). Degradation of LLVY was measured by incubating the enzyme sample with 13 μm LLVY in 0.1 M Tris buffer, pH 7.5 (assay volume, 1 ml) for 60 minutes at 37°C on a metabolic shaker. The reaction was stopped by the addition of 0.2 ml of ethanol on ice. The increase in fluorescence due to liberated AMC was measured on a Sequoia-Turner fluorometer with excitation and emission wavelengths of 390 and 440 nm, respectively. Data are expressed as nanomoles of AMC liberated per 60 minutes or fluorescence units per 60 minutes for column profiles.

Continuous monitoring of LLVY degradation was performed at an excitation wavelength of 380 nm (slit width, 15 nm) and an emission wavelength of 440 nm (slit width, 5 nm) at 37°C. The ammonium sulfate fraction (100 μl of a 1 : 10 dilution was incubated with 13 μm LLVY in Tris (2 ml of 100 mM total assay volume, pH 7.5), and the fluorescence due to liberation of AMC was monitored until a linear rate was established. Insulin at 10^"5M (220 μl of 10^"4M) was introduced into the cuvette, and the change in the rate of AMC liberated was compared with the introduction of an equal volume of buffer. Alternatively, repeated doses of 10^"6M insulin (20 μl of 10^"Vi) were introduced, and the rate was compared with the addition of equal volumes of buffer. Effect of peptide hormones on fluorogenic substrate degradation. The enzyme complex or the fully purified multicatalytic proteinase was incubated with and without various concentrations of insulin, ANP, relaxin, IGF-II, proinsulin, glucagon, EGF, or IGF-I, and the degradation of LLVY, LSTR, and LLE was measured. The effect of the peptide hormones on the amount of degradation by the complex of insulin degrading enzyme and multicatalytic proteinase was compared with their effects on degradation by purified multicatalytic proteinase. In addition, the effect of a variety of peptides and proteins, including insulin C-peptide, secretin, follicle-stimulating hormone (FSH), growth hormone, bovine serum albumin (BSA), tumor necrosis factor, α2 macroglobulin, calmodulin, bradykinin, bovine pancreatic polypeptide (BPP), ubiquitin, vasoactive intestinal peptide (VIP), and cholecystokinin (CCK) was measured and compared to the vehicle only.

Kinetics experiments. The degradation of LLVY and LSTR by the complex (ammonium sulfate fraction) at various substrate and insulin concentrations was measured. The LLVY or LSTR concentration was varied from 3 to 67 mmol/1, and the insulin concentration was set at 1.0 mmol/1, 10 mmol/1, 50 mmol/1, 0.1 μmol/l, 0.5 μmol/l, or 1.0 μmol/l. Dixon transformations were performed on the data. Background fluorescence in the absence of enzyme was subtracted at all concentrations. A similar experiment was performed with casein. LLVY degradation was measured as above with varying concentrations of α-casein (0, 2.1, 8.5, and 21 mmol/1). LLVY concentrations were 10, 50, and 100 mmol/1.

Results

The complex of insulin-degrading enzyme and multicatalytic proteinase was isolated and it was observed that insulin inhibits the chymotrypsin-like activity of the multicatalytic proteinase of the complex, as measured by the degradation of LLVY. Table 1 shows that insulin, a substrate for insulin-degrading enzyme, and LLVY, a substrate for multicatalytic proteinase, do not have direct effects on the other purified enzyme after separation from the complex. These data show that the effect of insulin on LLVY degradation requires an interaction between insulin- degrading enzyme and multicatalytic proteinase. TABLE 1. Effects of insulin and LLVY on activities of the complex of insulin degrading enzyme and multicatalytic proteinase and purified insulin- degrading enzyme and multicatalytic proteinase

Insulin-degrading LLVY-degrading activity activity complex of insulin 21 + 4 166 + 9 degrading enzyme and multicatalytic proteinase

Plus insulin ND 92 + 5

Plus LLVY 20 + 3 ND

Purified IDE 22 + 1 0

Plus LLVY 21 + 1 ND

Purified MCP 0 147 + 6

Plus insulin ND 141 + 3

The effect of 1 mmol/1 insulin on LLVY degradation and 40 mmol/1 LLVY on insulin degradation was measured. The degradation was carried out by the complex of insulin degrading enzyme and multicatalytic proteinase or by either purified insulin-degrading enzyme or multicatalytic proteinase, as described in METHODS. The insulin-degrading activity is expressed as a percentage of TCA-soluble counts from insulin per 15 minutes LLVY degradation is expressed as the fluorescence liberated (arbitrary units) per 60 minutes Data are means + SE from two independent experiments. ND, not determined.

Although insulin is the preferred substrate for insulin-degrading enzyme, other peptides can bind to this enzyme and act as substrates or inhibitors. Various of these were added to the IDE-MCP complex and separately to the purified multicatalytic proteinase, and LLVY degradation was measured. All of the peptides known to interact with insulin-degrading enzyme inhibited LLVY degradation by the complex, with insulin being the most effective (Fig. 1). The insulin-degrading enzyme substrates ANP and IGF-II, and relaxin, which has structural homology with insulin, inhibited the complex. Glucagon, which is bound and degraded by insulin- degrading enzyme, and EGF, which is bound but a poor substrate, had direct effects on purified multicatalytic proteinase as well as the complex. However, glucagon had a greater effect on the complex than on purified multicatalytic proteinase. Proinsulin, a competitive inhibitor of insulin degradation that is itself poorly degraded, had a partial effect. These data support a complex interaction between insulin-degrading enzyme binding and degradation and the regulation of multicatalytic proteinase.

Multicatalytic proteinase has proteolytic activity toward other substrates due to multiple active sites in the multicatalytic protein. It is known in the art that insulin has differential effects on the activities with chymotrypsin-like and trypsin- like activity (LLVY and LSTR degradation, respectively) being affected more than the peptidyl-glutamyl hydrolyzing activity (LLE degradation). Figures 2 and 3 show that the peptides that interact with insulin-degrading enzyme have effects on LSTR degradation similar to those on LLVY degradation but not on LLE degradation by the IDE-MCP complex. These data support the specificity of the insulin effect and of the interaction of IDE with MCP. These data also show that glucagon has effects on the complex separate from its direct effect on multicatalytic proteinase, since the hormone decreased LSTR degradation by the complex but not by purified multicatalytic proteinase. Also of interest is that insulin has a small but significant inhibition of LLE degradation. These data support a role for insulin-degrading enzyme regulation of multicatalytic proteinase activity and differential effects of insulin.

Numerous other peptides shown not to interact with insulin degrading enzyme have been examined in our system. These peptides include insulin C- peptide, secretin, FSH, growth hormone, BSA, TNF, 12 macroglobulin, calmodulin, bradykinin, BPP, ubiquitin, VIP, and CCK. These peptides did not significantly alter the proteolytic activity of the complex of insulin degrading enzyme and multicatalytic proteinase (data not shown). These data demonstrate the specificity of insulin-degrading enzyme substrates and show that the inhibition is not due to bulk peptide or vehicle effects.

In addition to insulin, glucagon, and proinsulin, the insulin-degrading enzyme substrates best characterized are IGF-I and IGF-II. IGF-I binds to insulin- degrading enzyme with a relatively high affinity, but is degraded slowly, similar to proinsulin, whereas IGF-II is degraded rapidly, analogous to insulin. Conclusion

The IDE-insulin-interaction alters the function of a cytoplasmic protein, the multicatalytic proteinase, in a manner consistent with the known biological actions of insulin. These biological actions include a decrease in cellular glucocorticoid action and the inhibition of cellular protein degradation.

Example 2 -- Active Insulin-Degrading Enzyme is Required for Insulin Mediated Inhibition of the Complex of Insulin Degrading Enzyme and

Multicatalytic Proteinase Insulin degrading enzyme is inhibited by certain known proteinase inhibitors at concentrations that do not affect relevant activities of the multicatalytic proteinase. These inhibitors were used to study the whether active insulin degrading enzyme is required for the observed insulin effect on the complex of insulin degrading enzyme and multicatalytic proteinase.

Materials and Methods

Methods and reagents were as described in Example 1 with the following exceptions. The enzyme preparation used was partially purified by ammonium sulfate fractionation by a procedure known in the art. The enzyme was dialyzed overnight against at least 20 volumes of sodium acetate pH 6.2, with either no addition, ImM EDTA, or ImM EGTA, with three changes. Acetate salts of the divalent cations were added at the concentrations indicated.

Results Insulin degradation by insulin-degrading enzyme is reduced by treatment with EDTA. The regulatory function of insulin-degrading enzyme is also affected by EDTA. EDTA treatment of the complex of insulin degrading enzyme and multicatalytic proteinase decreases the chymotrypsin-like activity of multicatalytic proteinase as reflected by decreased degradation of LLVY. Insulin and insulin- degrading enzyme regulate the trypsin-like (LSTR degradation) as well as the chymotrypsin-like (LLVY degradation) activity. EDTA treatment dramatically decreases LSTR degradation and eliminates the insulin effect.

To further explore the regulatory role of insulin-degrading enzyme in control of multicatalytic proteinase, selected inhibitors were examined (Table 2). The metalloproteinase inhibitor, phenanthroline, inhibited insulin-degrading enzyme and abolished the insulin effect on LLVY degradation similar to EDTA. NEM and bacitracin, known inhibitors of insulin-degrading enzyme, also blocked the insulin effect. PMSF, at low concentrations, had no appreciable effect. Table 2. The Effect of Various Inhibitors on the Complex of Insulin

Degrading Enzyme and Multicatalytic Proteinase Addition Concentration IDE (%) LLVY degradation

- insulin + insulin

None 100 100 51.3

1,10 phenanthroline 0.1 mM 44.4 73.1 76.2

1.0 mM 0.2 30.6 30.6

PMSF 0.1 mM 93.7 1 11.2 68.0

1.0 mM 85.3 1 16.1 62.7

NEM 0.01 mM 56.0 63.4 66.0

0.1 mM 13.4 24.7 18.6

Bacitracin lO μg/ml 59.9 71.3 52.0 lOO μg/ml 24.5 39.2 35.4

PCMB 0.05 mM 5.1 * *

0.5 mM 6.4 * *

EDTA 1.0 mM 52.4 33.7 28.8

10 mM 7.9 32.8 30.1

EGTA 1.0 mM 50.0 44.0 44.0

10 mM 5.7 31.0 29.6

* No LLVY Degradation.

The IDE-MCP preparation was prepared as in "Materials and Methods." Insulin and LLVY degradation were measured in the presence of the inhibitors or vehicle at the indicated concentration. The degradation of LLVY was determined in the presence of 1.0 μM insulin or vehicle only. Values are expressed as % of degrading activity in the presence of vehicle only. The data are the means of four independent experiments. Table 3 compares the effects of inhibitors of insulin-degrading enzyme on the proteolytic activity of the complex of insulin degrading enzyme and multicatalytic proteinase and on the corresponding activity of purified multicatalytic proteinase. Phenanthroline, NEM, and bacitracin inhibit LLVY degradation when multicatalytic proteinase is complexed with insulin-degrading enzyme, but these agents are ineffective on purified multicatalytic proteinase. These findings support a regulatory effect of insulin-degrading enzyme on multicatalytic proteinase activity.

TABLE 3. The Effect of Inhibitors of IDE on MCP Activity 1

Activity (% of Control) Additions IDE-MCP Purified MCP

None 100% 100%

Phenanthroline (0.2mM) 21% 95%

NEM (0.2mM) 3% 100%

Bacitracin (l .OmM) 10% 85%

Conclusion

Insulin degrading enzyme activity is required for the observed effect of insulin on the complex of insulin degrading enzyme and multicatalytic proteinase.

Example 3 -- Insulin Fragments Inhibit the Complex of Insulin Degrading Enzyme and Multicatalytic Proteinase

The results reported in Example 2 indicate that active insulin degrading enzyme is required for the insulin effect on the complex of the complex of insulin degrading enzyme and multicatalytic proteinase. This suggests that products of insulin degradation by insulin degrading enzyme may be responsible for the insulin effect in the complex.

Materials and Methods Methods and reagents were as described in Examples 1 and 2 with the following exceptions. To test the effect of insulin fragments, insulin was incubated with insulin-degrading enzyme, and the products were separated on a Sephadex G- 50 column. Products that eluted after intact insulin (a heterogeneous mixture of insulin fragments) were pooled and added back to the complex of insulin degrading enzyme and multicatalytic proteinase. The inhibition of LLVY degradation by both crude (ammonium sulfate purified) and purified complex was examined.

Results

As shown in Table 4, the insulin degradation products inhibited LLVY degradation more effectively than 10^"7 mol/1 insulin.

TABLE 4. The effect of insulin and insulin degradation products on multicatalytic proteinase activity

Enzyme sample No addition Plus insulin Plus products

Crude complex 100 66.2 44.2

Purified complex 100 63.2 37.5

Purified MCP 100 93.9 104.5

Data are %. Values are normalized to MCP activity with no additions. The degradation of LLVY-MCP at various stages of purification was measured in the presence of insulin (0.1 μmol/l) or insulin degradation products (unknown concentration) generated by insulin- degrading enzyme and then purified by Sephadex G-50 chromatography.

Since the previous experiment used a high but undetermined concentration of insulin degradation products and since some insulin fragments are insulin-degrading enzyme substrates, the experiment reported in Table 5 examined this more carefully.

Highly purified insulin-degrading enzyme was incubated for varying times with 5 x 10 ^s mol/1 insulin containing a trace amount of B26 iodoinsulin. The degradation of insulin was assessed by TCA precipitation. At the indicated times, the complex of insulin degrading enzyme and multicatalytic proteinase (ammonium sulfate preparation) was added along with either LLVY or LSTR. The degradation of these substrates was assayed after an additional 60 minutes (Table 5). TABLE 5. The effect of incubation time on the production of insulin degradation products and multicatalytic proteinase activity

LLVY LSTR TCA solubility of

Pre-incubation time degradation degradation B-26 iodoinsulin

No insulin (0) 160 142

0 117 100 0.2

5 1 10 118 3.0

15 130 123 12.3

30 134 1 17 22.1

Values are the fluorescence generated per hour (LLVY and LSTR degradation) or the percentage of TCA solubility. Insulin degradation products of various size and concentration were produced by predegrading insulin (50 nmol/1) with purified insulin- degrading enzyme for the indicated times. The enzyme complex containing IDE-MCP was then added, and the effect of predegraded insulin on LLVY degradation was determined. The degree of insulin degraded in the preincubation was approximated by TCA solubility.

As can be seen, 5 x 10^"8 mol/1 insulin, without preincubation, suppressed

LLVY degradation by 27% and LSTR degradation by 30%. After 5 minutes of exposure to purified insulin-degrading enzyme, the TCA solubility of the insulin was 3%, rising to 12.3% and 22.1% after 15 and 30 minutes, respectively. As has been shown in the art, TCA solubility significantly underestimates actual degradation since partially degraded insulin remains TCA perceptible. Based on high-performance liquid chromatography studies the actual loss of intact insulin is three- to fourfold greater than the production of TCA soluble fragments. Thus, degraded insulin comprised -10, 40, and 80% of the material at 5, 15, and 30 minutes, respectively. In spite of this, all time points showed the inhibition of LLVY and LSTR degradation, showing that degradation products of insulin also affect the activity of multicatalytic proteinase. The loss of inhibition by insulin and its products is approximated by the generation of TCA soluble radioactivity. This suggests that low-molecular-weight products that will not bind to insulin-degrading enzyme are ineffective.

Conclusion

Products of insulin degradation by insulin degrading enzyme inhibit the complex of insulin degrading enzyme and multicatalytic proteinase. Example 4 -- Insulin-Derived Peptides Inhibit the Complex of Insulin Degrading Enzyme and Multicatalytic Proteinase

The ability of insulin fragments to inhibit the complex of insulin degrading enzyme and multicatalytic proteinase was further investigated using synthetic peptides with sequences of fragments of insulin.

Materials and Methods

Methods and reagents were as described in Examples 1 and 2 with the following exceptions. Peptides with the sequences of amino acids 10-16 and amino acids 1 1-16 of the insulin B-chain were synthesized using standard methods of solid phase peptide synthesis. These peptide have the sequences HLVEALY and LVEALY, respectively.

Results

The effect of insulin, insulin analogs, and insulin-derived peptides were determined using assays for the chymotrypsin-like, trypsin-like, glutamyl- transferase, and protein degradation activities of the complex of insulin degrading enzyme and multicatalytic proteinase. Insulin and lys-pro insulin exhibited the highest level of inhibition of both the chymotrypsin-like and the trypsin-like activities (Figs. 4A and 4B). The peptides HVEALY and LVEALY inhibited at between 10^"6 and 10^"5 molar (Figs. 5 A and 5B). Insulin, insulin analogs, and each of the peptides showed no significant inhibition of the glutamyl-transferase activity of the complex of insulin degrading enzyme and multicatalytic proteinase. When measuring degradation of ¹²⁵I-insulin hydrolysis by the complex of insulin degrading enzyme and multicatalytic proteinase, insulin and its full-length analogs showed the strongest inhibition (Fig. 7). Once again, the peptides showed inhibition in the range of 10^'6 to 10⁵ molar. Conclusions

Several insulin analogs and the insulin-derived peptides HLVEALY and LVEALY each inhibit the chymotrypsin-like, trypsin-like, and general protein degradation activities of the complex of insulin degrading enzyme and multicatalytic proteinase.

Insulin-degrading enzyme cleaves insulin between residues B9-10, between residues B 10- 11 , and between residues B 16- 17. The B 10- 16 (HLVEALY) peptide has insulin-like effects. The BI 1-B16 (LVEALY) peptide has effects at high concentrations. An insulin analog substituted at BIO (BIOASP) has less effect than insulin. An insulin analog substituted at B 16-B 17 (EQF) has no effect.

Example 5 — In Intact Cells, Insulin Inhibits the Complex of Insulin Degrading Enzyme and Multicatalytic Proteinase

The present study provides evidence that the major effect of insulin on cellular protein degradation is due to an effect on proteasome activity.

Materials and Methods

Methods and reagents were as described in Examples 1 and 2 with the following exceptions. The HepG2 cell line was a gift of D. Clemens of the Omaha VAMC. Dulbecco's Modified Eagle's Medium (DMEM) was from Life

Technologies (Grand Island, NY). Fetal bovine serum was from Intergen Co.

(Purchase, NY). The fluorogenic substrate methoxysuccinyl-phe-leu-phe-7-amido-

4-trifluoromethyl coumarin (FLF) was from Enzyme Systems Products (Dublin,

CA). The calpain inhibitors N-acetyl-leu-leu-norleucinal (calpain inhibitor I, ALLN) and N-acetyl-leu-leu-methioninal (calpain inhibitor II, ALLM) were from

Sigma (St. Louis, MO).

Peptide degeneration by partially purified multicatalytic proteinase.

Multicatalytic proteinase (proteasome) was partially purified from rat skeletal muscle cytosol by methods known in the art. The enzyme was incubated in Tris buffer (0.1 M, pH 7.5) with FLF (13_μM final for 60 minutes at 37°C with the indicated concentration of insulin. The reaction was stopped with ice cold ethanol, and the fluorescence due to the liberation of 7-amido-4-trifluoromethyl coumarin was measured at excitation and emission wavelengths of 390 nm and 515 nm, respectively.

Cell culture and peptide degradation in intact cells. Human hepatoma (HepG2) cells were maintained in DMEM supplemented with 10% fetal bovine serum in a 5% CO₂/95% air environment. For peptide degradation assays, subconfluent cultures were serum deprived overnight (18 hours) prior to treatment. Peptide degradation was assessed with the membrane permeable substrate (FLF) by modification of a previously published method. After hormone and/or inhibitor treatment, FLF was added to the cells to a final concentration of 13 μM and incubated one hour. The DMSO concentration from the addition of FLF did not exceed 0.04%. The cells were then disrupted by sonication, and the resulting cell medium/lysate was read in a fluorometer as above.

Inhibitor studies on cellular peptide degradation. The effect of protease inhibitors on FLF degradation by HepG2 cells was examined by treatment of the cells for two hours with inhibitor prior to insulin and/or FLF addition as described above. The calpain inhibitors I and II (ALLN and ALLM, respectively), were prepared from stock solutions in DMSO. The DMSO concentration from the addition of inhibitors die not exceed 0.15%.

Results

Insulin inhibited FLF degradation in a dose-dependent manner, with a calculated EC₅₀ = 1.1 x 1 O^M. Therefore, FLF appears to behave much like LLVY as a chymotrypsin-like substrate for the proteasome in vitro. The intracellular degradation of FLF was examined in human hepatoma (HepG2) cells. To determine the specificity of FLF as a proteasome substrate, protease inhibitors were used. The calpain inhibitors ALLN and ALLM inhibit calpain and cathepsins at nanomolar concentrations, with similar potencies. Both ALLN and ALLM also are inhibitory toward the proteasome, but at micromolar concentrations, and with markedly different potencies. Consistent with FLF hydrolysis catalyzed by the multicatalytic proteinase, ALLN inhibited FLF degradation in the micromolar concentration range, while ALLM had much less effect. These results confirm that the majority of FLF degradation in HepG2 cells is due to the proteasome.

The effect of insulin on proteasome activity in intact cells was examined. As shown in Fig. 7, insulin inhibited FLF degradation in a dose dependent manner, consistent with insulin's concentration dependent effect on total protein degradation in hapatomea cells. The calculated EC₅₀ fl0^'13M) is physiologically relevant. The maximal inhibition was 19%, consistent with the insulin effect on the suppression of proteolysis in other studies. Additional controls with the calpain inhibitors indicate that the observed insulin effect was on multicatalytic proteinase activity.

Conclusion

The present study was directed at examining a potential effect of insulin on proteasome activity in intact cells. Insulin inhibited FLF degradation by both isolated proteasomes and intact cells. The cellular inhibition was concentration- dependent with the concentrations required comparable with previous studies using transformed hepatocytes, which are highly sensitive to insulin. The magnitude of the inhibition is consistent with the known action of insulin on inhibition of protein degradation. This study supports a role for insulin control of proteasome activity in intact cells.

Example 6 -- In Intact Cells, Active IDE is Required for Insulin Mediated Inhibition of the Complex of Insulin Degrading Enzyme and Multicatalytic

Proteinase This study determines whether active insulin-degrading enzyme is required for insulin inhibition of protein degradation in intact cells by using an antibody that inhibits the enzyme.

Materials and Methods

Methods and reagents were as described in Examples 1 , 2, and 5 with the following exceptions. The proteasome inhibitor lactacystin was from E.J. Corey (Harvard University). Cellular Protein Degradation: Male Sprague-Dawley rats were fasted overnight and hepatocytes were isolated by a modification of the method of Terris and Steiner. Cells were resuspended in cell buffer at an approximate density of 10⁶ cells/ml, and incubated 30 minutes at 37°C. Cell viability was approximately 90%. Cellular protein degradation was measured by labeling cells with buffer containing H-leucine and 5X normal serum concentration of unlabeled amino acids (excluding leucine) for 60 minutes, then washing and chasing with 2mM unlabeled leucine. The cells were divided into three portions, and either 30μg/ml anti-IDE antibody C20-3.1a(3), nonspecific mouse IgG (30μg/ml), or PBS buffer was added. To each of these systems, the following were added: 5X amino acids, lOnM porcine insulin, or no addition. Five times the normal serum concentration of amino acids were used as a positive control to demonstrate the inhibition of protein degradation by mass action, independent of the insulin regulatory system. The cells were incubated at 37°C, and 0.5 ml aliquots were taken at 0 and 120 minutes, and counted. Cellular degradation was determined by the difference in solubility (in 12.5% TCA) at 0 and 120 minutes.

In Vitro Proteasome Activity: Partially purified insulin-degrading enzyme, complexed with the multicatalytic proteinase, was obtained from rat skeletal muscle by methods known in the art. IDE-proteinase complex was preincubated five minutes with increasing amounts of anti-IDE antibody C20-3.1A. Degradation of insulin ¹²⁵I-labeled at the A14 position was measured by the generation of trichloroacetic acid soluble counts. Proteasome activity was measured with LLVY and LSTR for determination of chymotrypsin-like and trypsin-like activities, respectively. Antibody Studies: Subconfluent cultures of HepG2 cells were serum- deprived overnight, then osmotically loaded with the inhibitory anti-IDE monoclonal antibody C20-3.1A (50μg/ml) by the method of Okada and Rechsteiner. The cells were allowed to recover for one hour. The fluorogenic proteasome substrate FLF was added, and the cells were incubated for one hour. Fluorescence due to degradation of FLF was measured as described hereinabove. Results

The results reported in Table 6 show the intracellular insulin-IDE effect on total cellular protein degradation. Protein degradation was decreased by incubation with insulin or an excess of amino acids. Prelabeled cells were also incubated with a monoclonal antibody which inhibits insulin-degrading enzyme activity. Antibody- treated cells no longer responded to insulin by decreasing protein degradation but did respond to the addition of a fivefold excess of amino acids which work by mass action, independent of insulin. These data indicate a selective role for insulin- degrading enzyme in the cellular response to insulin for inhibition of protein degradation.

TABLE 6. Anti-IDE antibody eliminates inhibition of protein degradation in isolated rat hepatocytes.

Cellular protein degradation as measured by the release of radiolabeled amino acids is inhibited by insulin and excess amino acids in the buffer or a non-specific IgG. In the presence of anti-IDE antibody, however, insulin no longer inhibits protein degradation. The data are expressed as the percent soluble label released normalized to that in cells incubated without insulin or excess amino acids.

The data in Table 6 do not indicate the specific site of the insulin-IDE effect. Since we have shown an insulin-IDE-proteasome interaction, isolated proteasomes were incubated with varying amounts of anti-IDE antibody with and without insulin (Table 7). Two different catalytic sites of the proteasome, the trypsin-like and the chymotrypsin-like, were assayed using artificial substrates. As shown previously, insulin inhibited LLVY (chymotrypsin-like) and LSTR (trypsin-like) degradation. The anti-IDE antibody blocked the insulin effect on LLVY and LSTR degradation in a dose dependent manner. TABLE 7: Inhibition of IDE with a monoclonal antibody decreases insulin inhibition of the proteasome in vitro.

Insulin degradation was measured in the presence of increasing amounts of anti-IDE antibody. The data are expressed as the percent acid soluble counts per 15-minute incubation. Proteasome activities were measured under the same conditions in the presence or absence of lμM insulin. Data are expressed as the percent inhibition of proteasome activity compared with that in the absence of insulin. Data are mean _+ SEM for three independent experiments.

We also demonstrated that the in vivo effect of insulin also involved mediation by insulin-degrading enzyme by monitoring degradation of a fluorgenic substrate (FLF) by the multicatalytic proteinase in cultured hepatoma cells. These cells were osmotically loaded with insulin-degrading enzyme inhibitory monoclonal antibody or with vehicle only. After overnight incubation to allow recovery, insulin was added to the cells and FLF degradation assayed. In the cells loaded with vehicle only, insulin inhibited proteasome activity in a dose dependent manner as expected. Cells containing inhibitory antibody had a greatly diminished response to the hormone.

Conclusion

In this example, an antibody that inhibits the activity of insulin-degrading enzyme, was used to demonstrate that insulin-degrading enzyme is required for insulin inhibition of protein degradation in intact cells. The anti-IDE antibody blocked the insulin effect on cellular degradation of proteins prelabeled with radioactive amino acids. The anti-IDE antibody also decreased insulin inhibition of proteasome degradation of a specific substrate in intact cells. These data indicate that insulin works intracellularly via insulin-degrading enzyme to inhibit protein degradation by the proteasome. Example 7 -- In Intact Cells, Insulin-Derived Peptides Inhibit the Complex of Insulin Degrading Enzyme and Multicatalytic

Proteinase

The insulin-derived peptides HLVEALY and LVEALY were studied to determine an effect on the activity of the complex of insulin degrading enzyme and multicatalytic proteinase in intact isolated rat hepatocytes in primary culture and in a liver culture cell line, HepG2.

Materials and Methods Methods and reagents were as described in Examples 1, 2, 5, and 6.

Results

Insulin and both of the insulin-derived peptides, HLVEALY and LVEALY, inhibit complex of insulin degrading enzyme and multicatalytic proteinase FLF degradation in HepG2 cells (Figs. 8A and 8B). Inhibition by insulin is observed at least 120 minutes after exposure of the cells to that protein. The inhibitory effects of the peptides are transient. Inhibition is observed at 60 minutes after addition of a peptide, but not at 120 minutes after addition of a peptide. The effect of insulin on the peptides were also examined in isolated hepatocytes (Fig. 9). Inhibition by insulin was apparent for up to about 90 minutes. Inhibition by the peptide LVEALY at 10^"10 molar was apparent for at least 120 minutes (Fig 10A) . Inhibition by the peptide HLVEALY was transient, disappearing by 120 minutes after addition of the peptide (Fig 10B).

Conclusions

The insulin-derived peptides HLVEALY and LVEALY inhibit protease activity of the complex of insulin degrading enzyme and multicatalytic proteinase in intact cells. Example 8 -- In Intact Cells, Insulin, Insulin Analogs, and Insulin-Derived Peptides Inhibit Total Cell Protein Degradation

Total cell protein degradation was measured in the presence of insulin, insulin analogs, and insulin-derived peptides to determine whether the inhibition by these polypeptides of the complex of insulin degrading enzyme and multicatalytic proteinase had an effect of protein turnover in the cell.

Experiment 1

Materials and Methods Methods and reagents were as described in Examples 1, 2, 5, and 6 with the following exceptions. The hepatocyte cell line was H4, which was used in experimental studies as described hereinabove for the cell line HepG2.

Results In the experiment with results illustrated in Fig. 1 1, cells were labeled overnight, washed, and then protein degradation was allowed to proceed for three hours in the presence of various concentrations of insulin, insulin analog, or insulin- derived peptide. Insulin inhibited protein degradation at physiological concentrations of 10^""-10^"9 molar. The mutant insulin with an amino acid change in the B-chain at position 10 inhibited to a lesser degree than insulin. The peptide LVEALY showed no significant inhibition of protein degradation. The peptide HLVEALY caused significant inhibition of protein degradation at concentrations of 10^"9 to 10^"7 molar.

In another experiment with results illustrated in Fig. 12, cells were labeled overnight and washed, and preincubated for three hours. Inhibitor was then added and protein degradation was assessed at the end of a four-hour period. Insulin and the insulin analogs BIO and EQF inhibited protein degradation. The peptide LVEALY showed no significant inhibition. The peptide HLVEALY showed inhibition at 10 '° and 10 °. Conclusion

Insulin, insulin analogs, and an insulin-derived peptide effectively inhibit protein degradation in intact hepatocytes.

Experiment 2

Total cell protein degradation was measured in the presence of insulin and insulin-derived peptides under various conditions of labeling and exposure to peptides to determine whether these conditions affected inhibition by these polypeptides of protein turnover in the cell.

Materials and Methods

In this experiment cultured hepatocytes (H4 cells) were incubated with fH] leucine for varying times. Then unincorporated label was washed off the cells were incubated with excess unlabeled leucine. Cells were treated with varying concentrations of insulin or insulin derived peptide. Protein degradation was assayed by the production of acid soluble radioactivity as described hereinabove. Otherwise, methods and reagents were as described in Examples 1, 2, 5, and 6; the hepatocyte cell line was H4, which was used in experimental studies as described hereinabove for the cell line HepG2.

Results

The effect of insulin and the peptides on protein degradation under two different labeling conditions is shown in Figures 31 and 32. Figure 31 shows a dose dependent effect of insulin decreasing protein degradation under standard conditions (18 hours labeling, 3 hours wash, and 4 hour incubation). HLVEALY has a triphasic effect very similar to that seen with isolated proteasomes (see Example 4 and Figures 5 A and 5B above). LVEALY has a similar but less pronounced effect. Figure 32 shows insulin and peptide effects under different conditions of labeling and treatment (no wash, incubation medium without Ca⁺⁺ but with amino acids). Under these conditions both peptides decrease protein degradation at lower concentrations than insulin but with less total effect. Conclusion

Both peptides decrease protein degradation in cultured hepatocytes under both sets of conditions.

Example 9 -- In Animals, Insulin-Derived Peptides Inhibit Total Cell Protein Degradation Insulin derived peptides were evaluated for their ability to affect symptoms of a disorder of absolute or relative insulin deficiency or severe insulin resistance and of protein catabolism in rats.

Materials and Methods

Diabetic Sprague-Dawley rats were divided into treatment and control groups. The rats were dosed with either vehicle or insulin derived peptide by continuous infusion from an implanted osmotic pump. Doses of peptide, in μg/hr are shown in Figures 13-16. The animals were evaluated for urine excretion and body composition by methods known in the art.

Results In the first experiment, untreated diabetic rats (n=l) were compared to insulin-treated (n=2) and HLVEALY peptide-treated (n=l) rats. The results are reported in Figures 13 and 14. HLVEALY peptide at doses of 0.5 μg/hr and at 1 μg/hr had significant and consistent effects on urine output, body weight, and weights of various muscles and organs. These data show clear biological effects in decreasing the symptoms of a disorder of absolute or relative insulin deficiency or severe insulin resistance and of protein catabolism in rats. In these studies, the peptide actually reduced the size of fat pads and skeletal muscles. The peptide had no effects on glucose levels.

In a second experiment insulin-treated diabetic rats (n=l) were compared to rats treated with both insulin and HLVEALY peptide (n=l). The results are reported in Figures 15 and 16. Again, HLVEALY had significant effects of urine volume and weights of body and organs. At doses of 5 μg/hr and 8 μg/hr HLVEALY increased weights of fat pads and skeletal muscle over untreated and insulin-treated diabetic controls. The peptide had no effects on glucose levels.

Conclusion

These data show significant biological effects of the HLVEALY peptide on symptoms of a disorder of absolute or relative insulin deficiency or severe insulin resistance and of protein catabolism in a relevant animal model.

Example 10 -- In Diabetic Animals, Insulin-Derived

Peptides Preserve Muscle Mass at the Expense of Fat Stores

Insulin derived peptides were evaluated for their ability to affect symptoms of a disorder of absolute or relative insulin deficiency or severe insulin resistance and of protein catabolism in rats.

Materials and Methods

Rats were made diabetic by treatment with streptozotocin and implanted with subcutaneous osmotic minipumps. The minipumps administered either buffer, HLVEALY at 0.1 to 8.0 μg per hour, LVEALY at 0.05 to 5.0 μg per hour, or a combination of these two peptides at 0.1 to 2.5 μg per hour for each peptide. Insulin was administered either by mini pump (regular insulin, 2.4 U/da7) or by subcutaneous injection (PZI insulin at 1.5 to 3.5 units per day).

Blood glucose levels and urine 3-methylhistidine levels were measured daily. After 6 days the animals were killed and organ weights measured. Blood was obtained for assay of various metabolites.

Results

Figures 17 and 18 shows initial and final blood glucose levels, respectively. Insulin treatment significantly reduced glucose levels. The peptides produced slight, but not significant, reductions in glucose. Figure 19 shows urinary N-methylhistidine excretion. N-methyl histidine levels are known in the art to correlate with levels of muscle breakdown. This measurement of Figure 19 demonstrates that muscle breakdown was increased in diabetes and reduced by insulin. HLVEALY was as effective as insulin in decreasing muscle catabolism. LVEALY had no significant effect. Figures 20, 21, and 22 show serum levels of triglycerides, β- hydroxybutyrate, and non-esterified fatty acids, respectively. All were increased by diabetes and reduced by insulin treatment. HLVEALY restored triglycerides to non- diabetic levels with no significant effect on levels of β- hydroxybutyrate and non- esterified fatty acids. LVEALY had no significant effect on levels of any of these materials.

In these short term experiments no significant effects on body or organ weights were seen. In four of the five experiments (excluding one experiment in which the animals were less diabetic) HLVEALY showed some preservation of muscle weight and reduction of body fat (Figures 23-30). Figure 23 shows absolute epitrocharis muscle weight in control animals and rats treated with insulin, HLVEALY, and LVEALY. Figure 24 illustrates the effect on epitrocharis muscle weight in control and experimental animals expressed as % of total body weight. The results in Figures 23 and 24 demonstrate that LVEALY actually reduced the weight of this metabolically active muscle but HLVEALY tended to increase it. Epididymal fat pads, however, tended to be even lower in HLVEALY treated animals than in untreated diabetics (Figures 25 and 26). Similar trends were seen in fact pad ratios to whole leg, solus, and epitrocharis (Figures 27-29). Total body weight of HLVEALY treated animals tended to be lower (Figure 30).

Conclusion

HLVEALY treatment decreased muscle breakdown in diabetic rats. This treatment tended to preserve muscle at the expense of fat stores. An effect on fat turnover was supported by the reduction in serum triglycerides in HLVEALY treated rats. These data show significant biological effects of the HLVEALY peptide on symptoms of a disorder of absolute or relative insulin deficiency or severe insulin resistance and of protein catabolism in a relevant animal model.

Example 11 -- In Intact Cells, Insulin and Insulin-Derived Peptides

Decrease Protein Degradation and Lipid Synthesis; and Increase Glucose Transport, Glucose Oxidation, and IL-8 Secretion; but Insulin-Derived Peptides Do Not Affect DNA Synthesis

Several features of the cellular insulin response were measured in the presence of insulin and insulin-derived peptides to determine whether these polypeptides had an effect on protein degradation, lipid synthesis, glucose transport or oxidation, IL-8 secretion, and DNA synthesis in intact cells.

Experiment 1 Accepted models for insulin action, such as those described hereinabove, include a role for insulin processing and degradation in fat deposition and metabolism as well as protein turnover. This study examines insulin and peptide effects on isolated fat cell metabolism.

Materials and Methods

Adipocyte cells were obtained and cultured by methods described hereinbelow and by methods common in the art.. Otherwise, reagents and methods were generally as described in Examples 1, 2, 5, and 6. Glucose metabolism was measured by methods known to those of skill in the art.

Results and Discussion

The effects of the peptides on intracellular glucose metabolism in adipocytes are shown in Figures 33 and 34.

Insulin is known to increase glucose transport in fat cells. This is an effect of insulin that occurs rapidly upon exposure to insulin, that is not altered by processing of insulin, and, thus, was not included in the present study. Subsequently glucose can be stored as fat, an intermediate effect, or oxidized.

In intact adipocytes, HLVEALY decreased incorporation of glucose into lipid (Figure 33) and increased glucose oxidation (Figure 34).

Conclusion

The effects of the insulin derived peptide of decreasing incorporation of glucose into lipid and increasing glucose oxidation are those expected based on the studies of diabetic rats reported herein. These effects are consistent with present knowledge of the effects of insulin.

This effect indicates that insulin derived peptides can have marked clinical benefit in obese, insulin resistant type 2 diabetes where fat storage is a primary problem.

Experiment 2

Insulin modulates many physiological pathways, even in cells not classically characterized as insulin-sensitive. Many of these pathways have intermediate responses to insulin, for example, as described hereinabove. Endothelial cells alter cytokine secretion in response to insulin, which serves as a modulator of primary stimulants.

Materials and Methods

Endothelial cells were obtained and cultured by methods common in the art. Otherwise, reagents and methods were generally as described in Examples 1, 2, 5, and 6. IL-8 stimulation was achieved and levels measured by methods known to those of skill in the art.

Results

The interaction among dexamethasone, insulin, and TNF and their effects on IL8 secretion from respiratory endothelial cells are shown in Figure 35. Insulin increased IL8 secretion in TNF stimulated cells. LVEALY (peptide D) has no significant effect on TNF-stimulated IL-8 secretion. HLVEALY (peptide B) has a greater effect than insulin on TNF-stimulated IL-8 secretion. Similarly, on cells treated with TNF plus dexamethasone, LVEALY has no significant effect, and HLVEALY shows a greater effect than insulin.

Conclusion

Again, a selective effect of insulin derived peptide mimicking a cellular action of insulin is apparent.

Experiment 3

Among the effects of insulin that are not related to insulin processing and degradation insulin stimulated DNA synthesis. Insulin-derived peptides were evaluated for their effect on DNA synthesis.

Materials and Methods

Methods and reagents were as described in Examples 1, 2, 5, and 6 with the following exceptions. The hepatocyte cell line was H4, which was used in experimental studies as described hereinabove for the cell line HepG2.

Results

Figure 36 shows results indicating that the insulin-derived peptides do not stimulate DNA synthesis in hepatocytes, but insulin does.

Conclusions The lack of effect of the insulin-derived peptides on DNA synthesis is expected based on the activities of these peptides as reported herein. These effects are consistent with present knowledge of the effects of insulin. This action of insulin does not depend on degradation of the insulin, and peptides that mimic degraded insulin should not stimulate DNA synthesis. Example 12 -- In Obese Type 2 Diabetic Rats Insulin-Derived Peptide Decreases Hyperglycemia and Muscle Breakdown

An insulin derived peptide was evaluated for its ability to affect symptoms of a disorder of absolute or relative insulin deficiency or severe insulin resistance and of protein catabolism in rats. Specifically, the in vivo data from diabetic rats and the in vitro fat cell studies indicate value for insulin-derived peptide in reducing symptoms of type 2 diabetes in obese subjects.

Experiment 1 Materials and Methods

Generally, materials and methods are as described above in Examples 1, 2, 5, 6, 9, and 10. The rats employed were Zucker fatty diabetic animals. Other procedures and reagents are as commonly employed in the art.

Results

Only two of the experimental animals started developing hyperglycemia over the course of the experiment.

Muscle breakdown was monitored by measuring levels of 3-methylhistidine excretion. The results are shown in Figure 37. The 3-methylhistidine levels increased progressively in the buffer treated rat, but decreased over the first three days in the HLVEALY treated animal. HLVEALY decreases muscle breakdown in a type 2 animal model as it does in insulin deficient rats.

The pattern achieved with HLVEALY administration, i.e., a decrease in 3- methylhistidine early and escape later, may be due to degradation of the peptide.

Conclusions

Insulin-derived peptide decreased muscle breakdown and hyperglycemia in obese type 2 diabetic animals. Experiment 2

Materials and Methods

Generally, materials and methods are as described above in Examples 1, 2, 5, 6, 9, and 10. The rats employed were Zucker fatty diabetic animals. Other procedures and reagents are as commonly employed in the art.

Adipocyte preparation. Zucker diabetic fatty rats (ZDF/Gmi^tm-fa/fa) were treated with (n=2)or without (n=2) insulin-derived peptide HLVEALY for 7 days at 2 μg/hour using Alzet Model 2001 mini-osmotic pumps. Epididymal fat pads were removed and adipocytes prepared by collagenase digestion. Krebs-Ringer/HEPES buffer, pH 7.4, containing 4% bovine serum albumin and 0.55 mM glucose was used in all isolation and incubation steps. In this procedure, 1 g minced adipose tissue was incubated with 2 ml of buffer containing 5 mg collagenase for 45 min at 37° with gentle shaking. The cells were then washed 2 times in buffer and filtered through polyester silk. The cells were then aliquoted to microfuge tubes for assay. Fatty acid oxidation assay. Fatty acid oxidation was assessed by conversion of [9,10-³H] palmitate to ³H₂O. Freshly isolated adipocyte cells (2 x lOVml) were incubated in MEM with 2% BSA and 0.1 mM palmitate (lμCi/ μmol) at 37°C for 5 hours with and without ImM KCN. Oxidation taking place in the presence of KCN (mitochondrial inhibitor) was taken to be peroxisomal in origin. The excess [9, 10- H] palmitate in the media was removed by precipitation (2x) with 5% trichloroacetic acid. The supernatant was transferred to a microfuge tube, placed in a scintillation vial with 0.5 ml unlabeled water, sealed, and incubated at 50°C for 18 hr. The water outside the microfuge tube was then added to scintillation fluid and counted on a beta counter. The ³H₂O equilibrium coefficient was determined separately by adding 10 μl of ³H₂O to 490 μl water in a microfuge tube and incubated as above. The equilibrium coefficient was then used to calculate the total amount of ³H₂O produced by the cells. Results

Figures 38 and 39 show the results of this study. As the Figure 38 shows, total fatty acid oxidation was increased in the cells from treated animals. Figure 39 shows that much of this increase came from stimulation of peroxisomal fatty acid oxidation.

Conclusions

These results indicate that HLVEALY treatment increases fatty acid oxidation by adipocytes. Thus, an insulin-derived peptide decreased another symptom of obese type 2 diabetic animals. These results agree with the study in Sprague-Dawley rats, described herein above, that showed a decrease in epidymal fat pad weight in treated animals.

The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

Claims

WE CLAIM:

1. A polypeptide comprising a sequence from insulin flanking a cleavage site for an insulin-degrading enzyme, wherein the polypeptide inhibits an activity of a complex of insulin degrading enzyme and multicatalytic proteinase.

2. The polypeptide of claim 1 , wherein the polypeptide has a sequence from insulin flanking a cleavage site for an insulin-degrading enzyme.

3. The polypeptide of claim 2, wherein the polypeptide comprises insulin B-chain residues 9,10, 11, 16, 17, or combinations thereof.

4. The polypeptide of claim 3, wherein the polypeptide comprises insulin residues B 11 - 16 or B 10- 16.

5. The polypeptide of claim 3, wherein the polypeptide has a sequence HLVEALY or LVEALY.

6. The polypeptide of claim 1, wherein the polypeptide comprises a sequence of product of the degradation of an insulin by insulin degrading enzyme or by the complex of insulin degrading enzyme and multicatalytic proteinase.

7. The polypeptide of claim 1 , wherein the insulin comprises native insulin or AspBlO insulin.

8. The polypeptide of claim 1 , wherein the activity of the complex of insulin degrading enzyme and multicatalytic proteinase comprises hydrolysis of a substrate comprising the sequences LLVY or of LSTR adjacent to the cleavage site.

9. The polypeptide of claim 8, wherein the polypeptide does not significantly inhibit the LLE activity of the complex of insulin degrading enzyme and multicatalytic proteinase at a concentration where the polypeptide significantly inhibits hydrolysis of a substrate comprising the sequences LLVY or of LSTR adjacent to the cleavage site.

10. The polypeptide of claim 1, wherein the peptide is effective for reducing symptoms of a disorder of absolute or relative insulin deficiency, of severe insulin resistance, of lipid accumulation or excess lipid synthesis, or of protein catabolism or degradation upon administration to a subject in need thereof.

1 1. The polypeptide of claim 10, wherein the disorder comprises diabetes, severe stress, myocardial infarction, or a chronic wasting disease.

12. The polypeptide of claim 1 1 , wherein the severe stress comprises trauma, a burn, or starvation.

13. The polypeptide of claim 11 , wherein the chronic wasting disease comprises AIDS or cancer.

14. The polypeptide of claim 10, wherein the symptom comprises loss of muscle mass, increased body fat, increased lipid synthesis, or a combination thereof.

15. A method of detecting a disorder of absolute or relative insulin deficiency or severe insulin resistance in a patient comprising the steps of: obtaining a biological sample derived from the patient; measuring, in the biological sample, a level of an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and correlating the measured level with a known or predicted control level of a subject with certain characteristics of the patient or with a known or predicted level of a control group.

16. A method of detecting a disorder of protein degradation or catabolism in a patient comprising the steps of: obtaining a biological sample derived from the patient; measuring, in the biological sample, a level of an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and correlating the measured level with a known or predicted control level of a subject with certain characteristics of the patient or with a known or predicted level of a control group.

17. A method of detecting a disorder of lipid accumulation or excess lipid synthesis in a patient comprising the steps of: obtaining a biological sample derived from the patient; measuring, in the biological sample, a level of an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and correlating the measured level with a known or predicted control level of a subject with certain characteristics of the patient or with a known or predicted level of a control group.

18. A method of assessing the effectiveness of a treatment for absolute or relative insulin deficiency or severe insulin resistance comprising the steps of: obtaining a biological sample from a patient; measuring, in the biological sample, a level of an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and correlating the measured level with a prior level of an activity of insulin degrading enzyme or of the complex of insulin degrading enzyme and multicatalytic proteinase in a corresponding biological sample from the patient.

19. A method of assessing the effectiveness of a treatment for a disorder of protein degradation or catabolism comprising the steps of: obtaining a biological sample from a patient; measuring, in the biological sample, a level of an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and correlating the measured level with a prior level of an activity of insulin degrading enzyme or of the complex of insulin degrading enzyme and multicatalytic proteinase in a corresponding biological sample from the patient.

20. A method of assessing the effectiveness of a treatment for a disorder of lipid accumulation or excess lipid synthesis comprising the steps of: obtaining a biological sample from a patient; measuring, in the biological sample, a level of an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and correlating the measured level with a prior level of an activity of insulin degrading enzyme or of the complex of insulin degrading enzyme and multicatalytic proteinase in a corresponding biological sample from the patient.

21. A method of reducing symptoms of a disorder of absolute or relative insulin deficiency, of severe insulin resistance, of lipid accumulation or excess lipid synthesis, or of protein catabolism or degradation in a patient in need thereof, comprising the steps of: determining the need for reducing symptoms of a disorder of absolute or relative insulin deficiency, of severe insulin resistance, of lipid accumulation or excess lipid synthesis, or of protein catabolism or degradation; administering to the patient an effective amount of a polypeptide comprising a sequence flanking an insulin-degrading enzyme cleavage site of insulin; and monitoring the efficacy of the administration.

22. The method of claim 21 , wherein the disorder comprises diabetes, severe stress, myocardial infarction, or a chronic wasting disease.

23. The method of claim 22, wherein the severe stress comprises trauma, a burn, or starvation.

24. The method of claim 22, wherein the chronic wasting disease comprises AIDS or cancer.

25. The method of claim 21, wherein the symptom comprises loss of muscle mass, increased body fat, increased lipid synthesis, or a combination thereof.

26. The method of claim 21 , further comprising the step of measuring a level of an activity of the complex of insulin degrading enzyme and multicatalytic proteinase.

27. The method of claim 21 , further comprising the step of measuring a level of protein catabolism.

28. The method of claim 27, further comprising the step of measuring a level of degraded protein.

29. The method of claim 21 , wherein the polypeptide comprises a sequence from insulin flanking an insulin-degrading enzyme cleavage site of insulin, wherein the polypeptide inhibits an activity of a complex of insulin degrading enzyme and multicatalytic proteinase.

30. The method of claim 29, wherein the polypeptide comprises insulin B-chain residues 9, 10, 1 1, 16, 17, or combinations thereof.

31. The method of claim 30 wherein the polypeptide comprises insulin residues BI 1-16 or B 10-16.

32. The method of claim 30, wherein the polypeptide has a sequence of HLVEALY or LVEALY.

33. The method of claim 29, wherein the polypeptide comprises a product of the degradation of an insulin by insulin degrading enzyme.

34. The method of claim 21 , wherein the insulin comprises native insulin or AspB 10 insulin.

35. The method of claim 21 , wherein the activity of the complex of insulin degrading enzyme and multicatalytic proteinase comprises hydrolysis of a substrate comprising the sequences LLVY or of LSTR adjacent to the cleavage site.

36. The method of claim 35, wherein the polypeptide does not significantly inhibit the LLE activity of the complex of insulin degrading enzyme and multicatalytic proteinase at a concentration where the polypeptide significantly inhibits hydrolysis of a substrate comprising the sequences LLVY or of LSTR adjacent to the cleavage site.

37. A method of reducing symptoms of a disorder of absolute or relative insulin deficiency, of severe insulin resistance, of lipid accumulation or excess lipid synthesis, or of protein catabolism or degradation in need thereof comprising the steps of: determining the need for reducing symptoms of a disorder of absolute or relative insulin deficiency, of severe insulin resistance, of lipid accumulation or excess lipid synthesis, or of protein catabolism or degradation in need thereof comprising the steps of: administering to the patient an effective amount of a polypeptide comprising a sequence from atrial naturetic peptide, relaxin, or insulin-like growth factor II, wherein the polypeptide inhibits an activity of a complex of insulin degrading enzyme and multicatalytic proteinase; and monitoring the efficacy of the administration.