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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 (155, 205, and
26S)
and different pathways (ATP-dependent, ubiquitin, and non-ATP-dependent).
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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 activiy is
important for
ubiquitin-mediated proteolysis, insulin-altered proteolysis, antigen
processing,
apoptosis, cell growth and differentiation, and many other proteolysis-
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.
1 S 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 Znz+ but without the typical Znz+
binding
site. Insulin-degrading enzyme has a molecular weight of about 110,000
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
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be developmentally 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
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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 LLVY degradation by the complex of insulin degrading
enzyme and multicatalytic proteinase. The enzyme complex {D) or purified
multicatalytic proteinase (1) was incubated in the presence and absence of 1
Eunol/1
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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
S 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 (0) or
purified multicatalytic proteinase (~) were incubated,in the presence and
absence of
1 llrrlol/1 peptide hormones. The effect of the peptide hormone on the
degradation
by the complex versus purified multicatalytic proteinase was compared
(*P<p.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 (0) or purified multicatalytic
proteinase (~) were incubated in the presence and absence of 1 plnol/1 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 4A 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. B 10-asp inhibits less, and EQF shows
no
significant inhibition.
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Figures SA and SB illustrate dose response curies 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'sM. LVEALY
inhibits at 10-sM.
Figure 6 illustrates dose response effects for inhibition of degradation
of'ZSI
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'sM.
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 ECs° is 1.5 x 10'"M insulin, with a maximal inhibition
19.1 %.
**Indicates that the results are significantly different from no insulin added
(p<0.01 ).
Figures 8A 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 l0A and l OB 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 11 illustrates inhibition of total cell protein degradation in H4 cells
by
insulin, an insulin analog, and an insulin-derived peptide. Cells were labeled
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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 (3-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.
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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 Bolus 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.
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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
1 S 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
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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
10 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
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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
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20 of the A-chain and position 19 of the B-chain, and a third between
positions 6
and 11 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, Val, 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,
Gln may be replaced with Asp or Glu. In particular, AsnAl8, AsnA2l, or AspB3,
or
any combination of those residues, may be replaced by Asp or Glu. Also, G1nA15
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 B3, 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
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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: 1 ) and
LVEALY (SEQ ID NO: 2). These preferred polypeptides represent amino acids 10-
16 and I 1-16, 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
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example, peptides representing residues 1-9, 5-9, 1-10, 7-10, 9-24, 9-25, 10-
24, 10-
25, 16-21, lb-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
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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
5 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
10 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,
15 Asp, Asn and Gln (representing carboxyl group containing side chains):
Class IV:
His, Arg and Lys (representing basic side chains); Class V: Ile, Val, Leu, Phe
and
Met (representing hydrophobic side chains); Class VI: Phe, Trp, Tyr and His
(representing aromatic side chains); and Class VII: Lys, Asp, Giu, Asn and
Gln.
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
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16
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 ai.,
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
Gln; the exchange of Ser with Asn, Phe or Trp; the exchange of Leu with Tyr or
Trp;
and the exchange of Pro with Glu, Gln 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, TGFa, 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 I S 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, TGFa, 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.
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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 Garners 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
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18
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
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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%,
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
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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 S mg to 5000 mg of active ingredient, preferably 50
mg to
5 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
10 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
15 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
20 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.
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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,
1 S 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
['ZSI]iodoinsulin, specifically labeled on Tyl'"4 or Tyrezb, 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 j3-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.
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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 A14
yzsl]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 nm (LLE). Degradation of
LLVY was measured by incubating the enzyme sample with 13 N.m LLVY in 0.1 tv~
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, S nm) at 37°C. The ammonium sulfate fraction (100 Itl of a
1:10 dilution
was incubated with 13 pm LLVY in Tris (2 ml of 100 mtvt total assay volume, pH
7.5), and the fluorescence due to liberation of AMC was monitored until a
linear rate
was established. Insulin at l0~siv1 (220 pl of 10-0tn) 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 1 O~tvi insulin
(20 p.l of
10'°n~) were introduced, and the rate was compared with the addition of
equal
volumes of buffer.
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Effect of peptide hormones on fluoro enic 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, a2 macroglobuIin, 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/l, 10 mmol/1, 50 mmol/1, O.1
Nxrlol/l,
0.5 N.mol/1, or 1.0 Nxrlol/1. 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 a-casein (0,
2.1,
8.5, and 21 mmol/1). LLVY concentrations were 10, 50, and 100 mmol/l.
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.
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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 _+ S
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
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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
5 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
10 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
15 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-
20 peptide, secretin, FSH, growth hormone, BSA, TNF, I2 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
25 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.
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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, 1 mM EDTA, or 1 mM 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
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27
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 S 1.3
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 93.7 111.2 68.0
1.0 mM 85.3 116.1 62.7
NEM 0.01 mM 56.0 63.4 66.0
0.1 mM 13.4 24.7 18.6
Bacitracin 10 p,g/ml 59.9 71.3 52.0
100 pg/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
pM 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.
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Table 3 compares the effects of inhibitors of insulin-degrading enz~~me 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 LLVY Degrading
Activity (% of Control)
Additions IDE-MCP Purified MCP
None 100% 100%
Phenanthroline (0.2mM) 21 % 95%
NEM (0.2mM) 3% 100%
Bacitracin (I.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-
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29
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-' mol/1 insulin.
TABLE 4. The effect of insulin and insulin degradation products on
multicatalytic
proteinase activity
Enzyme sample No addition Plus insulinPlus products
Crude complex 100 66.2 44.2
Purified complex100 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
pmol/I) or insulin degradation products (unknown concentration) generated by
insulin-
degrading enzyme and then purified by Sephadex G-SO 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
S x
10-8 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).
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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 1 I 7 100 0.2
110 118 3.0
15 130 123 12.3
30 134 117 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
5 concentration were produced by predegrading insulin (50 nmol/I) 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.
10 As can be seen, S 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 signif cantly underestimates actual
15 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, 1 S, and 30
minutes, respectively. In spite of this, all time points showed the inhibition
of
20 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.
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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 l and 2 with the
following exceptions. Peptides with the sequences of amino acids 10-16 and
amino
acids 11-16 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'~ and 10-5 molar (Figs. SA and SB). 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'z5I-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~ to 10-5 molar.
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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 B10-11, and between residues B16-17. The BIO-16 (HLVEALY) peptide
has insulin-like effects. The B 11-B 16 (LVEALY) peptide has effects at high
concentrations. An insulin analog substituted at B 10 (B 1 OASP) has less
effect than
insulin. An insulin analog substituted at B16-B17 (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
1 S 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 (O.1M, pH 7.5) with FLF (13NM final for 60 minutes at 37°C with
the
indicated concentration of insulin. The reaction was stopped with ice cold
ethanol,
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JJ
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% COz/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 ~M 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 ECS° = 1.1 x 10'~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,
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3a
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 ECS° ('10-"M) 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).
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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 1 OG
cells/ml, and incubated 30 minutes at 37°C. Cell viability was
approximately 90%.
5 Cellular protein degradation was measured by labeling cells with buffer
containing
;H-leucine and SX 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 30pg/ml anti-IDE
antibody
C20-3.1 a(3), nonspecific mouse IgG (30pg/ml), or PBS buffer was added. To
each
10 of these systems, the following were added: SX amino acids, 1 OnM 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
15 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
20 minutes with increasing amounts of anti-IDE antibody C20-3.1 A. Degradation
of
insulin'ZSI-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.
25 Antibody Studies: Subconfluent cultures of HepG2 cells were serum-
deprived overnight, then osmotically loaded with the inhibitory anti-IDE
monoclonal antibody C20-3.1A (SOltg/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
30 due to degradation of FLF was measured as described hereinabove.
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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.
No antibody Anti-IDE nonspecific IgG
Control 100 100 I 00
SX amino acids 78.8 _+ 4.6 85.0 _+ 2.4 78.4
+ 6.5
Insulin 83.5 + 6.4 99.7 + 2.5 _
78.2 + 6.2
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.
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TABLE 7: Inhibition of IDE with a monoclonal antibody decreases insulin
inhibition of the proteasome in vitro.
insulin %
inhibition
by 1 uM insulin
p.g/ml Antibodydegradation
LLVY LSTR
0 100 64.5_+4.9 69.9
+2.9
20 94.6+g,5 45.8+8.7 _
52.6
+7.3
40 80.9_+10.7 45.2 _
+4.6 38.8
+13.7
60 68.2+9,g _ _
37.2+3.9 47.0+9.4
lnsufin degradation was measured in the presence of increasing amounts of anti-
IDE
antibody. The data are expressed as the percent acid soluble counts per I S-
minute
incubation. Proteasome activities were measured under the same conditions in
the presence
or absence of 1 pM 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.
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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-'° molar was apparent for at least 120 minutes (Fig l0A) .
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.
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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 l, 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 I, 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-' 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 B 10 and EQF inhibited protein degradation. The peptide
LVEALY showed no significant inhibition. The peptide HLVEALY showed
inhibition at 10-'° and 10-°.
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Conclusion
Insulin, insulin analogs, and an insulin-derived peptide effectively inhibit
protein degradation in intact hepatocytes.
5 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 ['H]
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 SA and SB 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.
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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
~,g/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=1) were compared to
insulin-treated (n=2) and HLVEALY peptide-treated (n=1 ) rats. The results are
reported in Figures 13 and 14. HLVEALY peptide at doses of 0.5 p,g/hr and at 1
ug/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=I ) were compared to
rats treated with both insulin and HLVEALY peptide (n=I ). The results are
reported
in Figures 15 and 16. Again, HLVEALY had significant effects of urine volume
and
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weights of body and organs. At doses of 5 ug/hr and 8 pg/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 p,g per hour, LVEALY at 0.05 to 5.0 p,g per hour, or a
combination of these two peptides at 0.1 to 2.5 ~g per hour fox 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.
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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, (3-
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 Vii- 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.
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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
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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).
5
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
10 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.
15 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
20 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,
25 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
30 IL8 secretion from respiratory endothelial cells are shown in Figure 35.
Insulin
increased IL8 secretion in TNF stimulated cells. LVEALY (peptide D) has no
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46
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.
S
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.
1 S 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.
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47
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.
I S 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.
C'nnrl»cinne
Insulin-derived peptide decreased muscle breakdown and hyperglycemia in
obese type 2 diabetic animals.
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48
Experiment 2
Materials and Methods
Generally, materials and methods are as described above in Examples l, 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'"'-fa/fa) were
treated with (n=2)or without (n=2) insulin-derived peptide HLVEALY for 7 days
at
2 pg/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 S 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-;H] palmitate to 3H2O. Freshly isolated adipocyte cells (2 x 106/ml)
were
incubated in MEM with 2% BSA and 0.1 mM palmitate (l~tCi/ Etmol) at
37°C for 5
hours with and without 1 mM KCN. Oxidation taking place in the presence of KCN
(mitochondria) 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'H20 equilibrium coefficient was determined
separately by adding 10 ~tl of ~HzO to 490 pl water in a microfuge tube and
incubated as above. The equilibrium coefficient was then used to calculate the
total
amount of'HZO produced by the cells.
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49
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.
C'nnrlneinne
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.
