Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR IDENTIFICATION OF COMPOUNDS STIMULATING INSULIN
SECRETION
TECHNICAL FIELD
According to the invention (3-tubulin, as well as a new polypeptide present in
pancreatic (3-
cells, have been identified as molecular targets for sulfonylurea compounds.
These findings
enable for the identification of new insulin secretagogues. The invention thus
relates to the
use of sulfonylurea compounds, such as e.g. glibenclamide, in methods for
identification of
to compounds binding the new polypeptide or tubulin, or stimulating tubulin
polymerization
and/or turnover, said compounds thereby stimulating insulin secretion.
BACKGROUND ART
Is
Diabetes mellitus is a chronic disease affecting approximately 3-5% of the
Swedish
population and it is associated with a variety of severe late complications
leading to
enhanced morbidity and mortality. Thus, this disease markedly compromises the
health and
life quality of affected individuals and consumes a substantial amount of the
health care
Zo budget.
Hyperglycemia is a major risk factor for the development of the specific
diabetes-associated complications and probably also for the increased risk of
cardiovascular disease. Early intervention, which requires diagnosis in the
prediabetic state,
Zs is expected to contribute to a reduction of the diabetic complications.
Hence, there is an
urgent need for precise methods in the early detection of individuals at risk
for the
development of diabetes. Furthermore, there is a need for the design of new
concepts and
drugs for prevention and treatment of diabetes. A better understanding of the
biochemical,
cellular, genetic and molecular basis for the development of hyperglycemia and
the effect
30 of hyperglycemia on both release and action of insulin will pave the way
for new drugs and
future development of gene therapy in the treatment of diabetes. A defective
insulin release
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PCT/SE00/01563
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is a characteristic of non-insulin dependent diabetes mellitus (NIDDM) and to
some extent
also to the early phase of insulin-dependent diabetes mellitus (IDDM).
An initial step in the [3-cell stimulus-secretion coupling is metabolism of
glucose, resulting
in the formation of ATP. ATP closes the ATP-regulated K+-channels (KATp
channels),
resulting in plasma membrane depolarization, opening of voltage-gated L-type
Ca2+-channels and increase in cytoplasmic free Ca2+ concentration, [Ca2+]i.
The KA1'p
channel is composed of at least two components: a sulfonylurea receptor (SUR)
and an
inward rectifier potassium channel protein (Aguilar-Bryan, L. et al. (1995)
Science 268:
io 423-426). The binding of sulfonylureas (SU) to SUR1, the (3-cell variant of
SUR, results in
the closure of the KpTp channels and thereby insulin release in the (3-cell.
Sulfonylurea
compounds, such as glibenclamide (glyburide; 5-Chloro-N-[2-[4-
[[[(cyclohexylamino)
carbonyl]amino]sulfonyl]phenyl]ethyl]-2-methoxybenzamide, CAS: [10238-21-
8])have
been used in the treatment of NIDDM (for a review, see Luzi, L. & Pozza, G.
(1997) Acta
is Diabetol. 34, 239-244).
Sulfonylurea compounds are capable of promoting insulin secretion even in the
absence of
changes in membrane potential and intracellular calcium. It has been shown
that
sulfonylurea compounds directly promote exocytosis of insulin (Eliasson L. et
al. (1996)
2o Science 271: 813-815; Flatt et al. (1994) Diabete et Metabolisme 20: 157-
162). This effect
is dependent on protein kinase C and is observed at therapeutic concentrations
of
sulfonylureas, which suggests that it contributes to their hypoglycemic action
in diabetics.
It has been suggested that 80-90% of the SU binding proteins is localized to
intracellular
membranes, including those of the secretory granules (Ozanne S.E. et al.
(1995)
z, Diabetologia 38, 277-282). The molecular mechanism underlying this direct
effect of
sulfonylureas on insulin exocytosis is not known.
Consequently, there is a need for identification of new intracellular targets
that could
explain the effect of sulfonylureas on insulin exocytosis via a pathway
independent of the
3o sulfonylurea receptor (SUR-1) and the ATP-regulated K+-channel . Such
targets could be
utilized in methods for the identification of new compounds stimulating
insulin secretion.
WO 01/13121 CA 02380983 2002-O1-23 PCT/SE00/01563
The involvement of the microtubular network in glucose-induced insulin
secretion has
been suggested (Lacy, P. E. et al. (1968) Nature 219, 1177-1179). The
hypothesis was
based on the decreased insulin secretion observed in the presence of drugs
that inhibit
polymerization of the tubulin heterodimer to form microtubules. Subsequent
studies
revealed that the reduced secretion was observed both in the presence of
microtubule
stabilizers (e.g. D20 and ethanol) and destabilizers (e.g. colchicine and
vincristine)
(Malaisse, W. J. et al. (1970) Diabetologia 6, 683; Malaisse, W. J. et al.
(1971) Diabetes
20, 257-265). It was also shown that the observed effect did not result from
an altered
~ o insulin production or calcium uptake, demonstrating that it was the
transport and/or
secretion of insulin containing granules that was not functional. Following
these results a
model of insulin secretion was presented in which the second sustained phase
of secretion
depends on the directional transport of insulin granules along the
microtubular network
(Malaisse, W. J. et al. (1974) Eur. J. Clin. Invest. 4, 313-318).
A number of reports have since confirmed the importance of the microtubular
system in
insulin secretion (for a review, see Howell, S. L. & Tyhurst, M. (1986)
Diabetes/Metabolism Reviews, 2, 107-123). These studies revealed that the
integrity and/or
the dynamic equilibrium (treadmilling as well as phases of microtubule growth
and
ao shortening) of the microtubules are essential for glucose-stimulated
insulin secretion. Of
interest from a pharmaceutical viewpoint is to identify agents that act on the
microtubule
network to specifically enhance insulin secretion. This task is not trivial as
the
microtubular network is generally involved in secretory processes in several
endocrine
organs and glands (see e.g. Poisner, A. M. & Bernstein, J. (1971) J.
Pharmacol. Exp. Ther.
as 177, 102-108; Neve, P. et al. (1970) Exp. Cell Res. 63, 457-460; Williams,
J. A. & Wolff,
J. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 1901-1908; Kraicer, J. & Milligan,
J. V. (1971)
Endocrinology 89, 408-412) and hence not expected to be a suitable target for
a specific
insulin secretagogue.
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BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1
Binding of [1251]glibenclamide to pancreatic (3-cells. (A) Binding of
glibenclamide was
s examined in intact (3-cells and subcellular fractions (cytosol and
membranes) derived
therefrom. Cross-linking (UV) did not affect the magnitude of the binding. (B)
The cross-
linked samples were further analyzed by SDS-PAGE and autoradiography (C,
cytosol and
M, membranes). These fractions were washed extensively, centrifuged and the
supernatant
(S) and pellets (P) further analyzed by SDS-PAGE and autoradiography. (C) The
cytosolic
io fraction was further analyzed by 2-dimensional electrophoresis and
autoradiography.
Fig. 2
Glibenclamide accelerates microtubule growth. (a): Experimental data showing
the
increase in optical density at 340 nm, resulting from tubulin polymerization
in vitro, as a
is function of time. Microtubule growth was induced by increasing the
temperature from
15°C (at t=0) to 35°C. The concentration of bovine brain tubulin
was 2.9 mg/ml and the
buffer solution contain 80 mM Pipes, 1 mM MgClz, 1 mM EGTA, 1 mM GTP, 2.1
DMSO and 10% glycerol at pH 6.9. The black symbols indicate the presence of
0.17 mM
glibenclamide whereas the gray symbols represent the controls in its absence.
The
zo polymerization was followed simultaneously for one pair at the time, with
each pair being a
glibenclamide sample and a control, to ensure comparisons are made on
identical protein
samples. The data presented is representative of four experiments. (b): The
rate of tubulin
polymerization as measured by the change in OD with time (dOD/dt) as a
function of time.
The experimental conditions were the same as in (a). Black lines indicate the
presence of
zs glibenclamide and gray lines its absence.
Fig. 3
Staining of microtubules with an antibody against tubulin in (3-cells
pretreated with
vehicle, nocodazole or taxol (a). Data are representative of three
determinations. Effect of
3o nocodazole (b) and taxol (c) on the dynamics of insulin release from column
perifused [3-
cells. Cells were incubated with 5 ~.M nocodazole for 30 min or 25 ~.M taxol
for 15 min
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prior to starting perifusion of the cells. Taxol was present during the whole
experiment.
The control cells were incubated with their respective vehicle solutions.
Protocols were
initiated at the times indicated in the figure with either 1 ~M glibenclamide
or 15 mM
glucose. The data represents the mean ~ SEM of four experiments.
DISCLOSURE OF THE INVENTION
According to the invention [3-tubulin, as well as a 65 kDa polypeptide present
in pancreatic
~o (3-cells, have surprisingly been identified as molecular targets for
sulfonylurea compounds.
These findings enable for the identification of new insulin secretagogues,
which can be
sulfonylureas or other compounds acting on these intracellular sulfonylurea
targets. The
methods according to the invention address a mechanism for insulin secretion,
which is
independent of the sulfonylurea receptor/K+-ATP complex.
is
To the inventors' knowledge, there are no indications in the prior art that
sulfonylureas
interact with tubulin or with the said -~-65 kDa polypeptide, which
polypeptide as such is an
aspect of the invention. Further, it has not been previously shown that
compounds which
stimulate insulin secretion also have a stimulating effect on tubulin
polymerization.
Consequently, in one aspect this invention provides the use of tubulin and/or
the ~65 kDa
polypeptide, identified according to the Examples below, as targets in the
identification of
active agents useful for stimulating insulin secretion and thereby useful in
the treatment of
hyperglycemia and diabetes, in particular NIDDM.
The said active agent could e.g. be a sulfonylurea derivative, or any other
compound acting
on intracellular targets indicated below. The said active agent could e.g. be
a derivative of a
"second generation" sulfonylurea, such as glibenclamide (glyburide) or
glipizide. In
particular, the said active agent should be stimulating insulin secretion
independently of the
3o plasma membrane sulfonylurea receptor activity in the presence, but not in
the absence, of
stimulatory glucose concentrations. As used herein the term "stimulatory
glucose
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concentrations" means a postprandial blood glucose level, such as blood
glucose higher
than approximately 8 mM.
This invention further provides a method for the identification of a compound
capable of
binding to the ~65 kDa polypeptide as defined in the Examples below, said
method
comprising the steps (a) contacting the said ~65 kDa polypeptide with a test
compound;
and (b) determining the binding of the said test compound to the said ~65 kDa
polypeptide.
Such a method could include the additional steps of contacting the said ~65
kDa
polypeptide with a reference compound binding to the said polypeptide; and
determining
~ o the binding of the said test compound, relative to that of the said
reference compound, to
the said ~65 kDa polypeptide.
When the method according to the invention involves the use of a reference
compound, the
reference compound could be any compound known to bind the 65 kDa polypeptide.
It is
Is shown in Example 1, below, that the sulfonylurea glibenclamide (glyburide)
is associated
with the 65 kDa polypeptide and thus glibenclamide is suitable as a reference
compound. It
is anticipated that additional suitable reference substances are other
sulfonylurea
compounds known to stimulate insulin secretion, such as tolbutamide,
chlorpropamide,
acetohexamide, tolazamide, glimepiride, or a "second generation" sulfonylurea
(like
Zo glibenclamide) such as glipizide. Sulfonylurea compounds that could be used
include those
having a generic formula disclosed in e.g. United States patent Nos.
3,454,635; 3,669,966;
or 4,379,785. However, by using the methods according to the invention, the
skilled person
will be able to identify additional sulfonylurea compounds, sulfonylurea
derivatives or
other substances, and subsequently use them as reference substances.
2s
In addition, the invention provides a method for the identification of a
compound capable
of binding to tubulin, comprising the steps (a) contacting tubulin with (i) a
test compound
and (ii) a reference compound which is a sulfonylurea compound, or a
derivative thereof,
binding to tubulin; and (b) determining the binding of the said test compound,
relative to
3o that of the said reference compound, to tubulin. The said sulfonylurea
compound could be
any of the sulfonylurea compounds discussed above. As mentioned above, by
using the
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methods according to the invention, the skilled person will be able to
identify additional
sulfonylurea compounds, sulfonylurea derivatives or other substances, and
subsequently
use them as reference substances.
In methods described above, binding, in particular a high degree of binding,
of the said test
compound to the ~65 kDa polypeptide or to tubulin, respectively, is indicative
of a
compound capable of stimulating insulin secretion.
An alternative method for the identification of a compound capable of binding
to tubulin,
~ o could comprise the steps (a) contacting tubulin with (i) a test compound
and (ii) a reference
compound which is a sulfonylurea compound, or a derivative thereof, binding to
tubulin;
and (b) determining the stimulating effect of the said test compound, relative
to that of the
said reference compound, to polymerization or turnover of tubulin. In such a
method, a
stimulating effect on polymerization or turnover of tubulin is indicative of a
compound
~ s capable of stimulating insulin secretion.
In the methods described above, the said tubulin could be from any source, but
is
preferably isolated from humans and in particular human (3-cells. Both a-
tubulin and [3-
tubulin consist of various isotypes and there are differences in their tissue
distributions
Zo (Roach, M.C. et al. (1998) Cell Motility and the Cytoskeleton 39, 273-285;
Luduena, R.F.
(1993) Mol. Biol. Cell 4:445-457; Luduena, R.F. (1998) Int. Rev. Cytology 178:
207-275).
The differences among the (3 isotypes are known to be conserved in evolution.
Since
isotypes may differ in their functional assignments or roles in cells, it may
be particularly
advantageous to use tubulin derived from (3-cells when screening for compounds
zs stimulating insulin secretion.
The reference compound could be labeled, e.g. radiolabeled or fluorescence
labeled, and
binding of the said reference compound to tubulin or the ~65 kDa polypeptide
could be
determined by methods such autoradiography or fluorescence spectroscopy (e.g.
3o fluorescence polarization, fluorescence correlation spectroscopy, or
confocal microscopy
(see e.g. Example 1, below)). For a general review on fluorescence
spectroscopy methods,
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see Joseph R. Lakowicz: Principles of fluorescence spectroscopy, Second
edition, Kluwer
Academic/Plenum Publishers, New York, 1999, ISBN 0-306-46093-9.
The polymerization of tubulin to form microtubules can be followed by a number
of
methods known in the art, e.g. by observing the increase in optical density of
a tubulin
solution as described in Example 2, below. There are numerous publications
available in
which this technique has been used (see e.g. Microtubule Proteins, Editor:
Avila, J., 1990,
CRC Press). Tubulin polymerization can also be followed using fluorescence
labeled
tubulin proteins, which upon polymerization end up in close proximity to each
other (e.g.
io quenching or fluorescence resonance energy transfer phenomena). As
mentioned above,
fluorescence spectroscopy methods are described in the art e.g. by Lakowicz,
supra.
Microtubule treadmilling (for a review, see Margolis, R. L. & Wilson, L.
(1998) Bioessays
20, 830-836) represents the simultaneous addition of a tubulin heterodimer at
one
1, microtubule end and the removal at the other. This phenomenon occurs in
living cells and
appears to be essential to the biological function of the microtubules
(Rodionov, V. I. et al.
(1997) Science 275, 215-218). The incorporation of fluorescence labeled
tubulin molecules
in pre-existing microtubules can thus be utilized in cell-based assays, or
alternatively in
vitro by fluorescence spectroscopy measurements, e.g. fluorescence anisotropy
or
Zo correlation spectroscopy, for screening of compounds that via the
microtubule network
affect secretion of insulin.
In a further aspect, this invention relates to a method for the treatment of
diabetes and/or
hyperglycemia comprising administering to a patient in need thereof an
effective amount of
zs a compound identified by any one of the methods according to the invention.
The term
"effective amount" means a dosage sufficient to provide treatment for the
disease state
being treated. The typical daily dose of the active compound varies within a
wide range and
will depend on various factors such as for example the individual requirement
of each
patient and the route of administration. In general, daily dosages could be in
the range of
30 0.1 to 1000 mg, such as 1 to 50 mg or 2.5 to 20 mg. It is anticipated that
the agents
identified by the methods according to the invention could have reduced side
effects
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compared to sulfonylurea compounds known in the art. For a review of the
adverse effects
and precautions of sulfonylurea drugs, see Paice, BJ et al. (1985) Adverse
Drug React.
Acute Poisoning Rev. 4: 23-36.
s In a further important aspect, this invention provides an isolated mammalian
polypeptide
characterized by binding to glibenclamide and an apparent molecular mass of
approximately 65 kDa. In particular, this polypeptide can be identified when
the cytosolic
fraction of pancreatic (3-cells from ob/ob mice is analyzed by SDS-PAGE.
However, it will
be understood that the invention includes the isolated glibenclamide-binding
~65 kDa
i o polypeptide from any mammalian source.
EXAMPLES
is EXAMPLE 1: Sulfonylureas associate with 47 kDa and 65 kDa proteins in (3-
cell cytosolic
fractions from ob/ob mice.
(a) Binding of glibenclamide
zo Ob/ob mice were obtained from a locally bread colony at the animal facility
within the
Karolinska Institute, Stockholm, Sweden. The ob/ob mice were starved
overnight. The
animals were decapitated, the pancreas was excised from the abdominal cavity
and
pancreatic (3-cells were isolated after collagenase digestion as previously
described
(Lernmark, A, (1974) Diabetologica 10, 431-438: Nilsson, T. et al. (1987)
Biochem. J. 248,
z, 329-336). The cells were kept overnight in RPMI 1640 medium supplemented
with 11 mM
glucose, 10% FCS, 100 ~,g/ml streptomycin and 100 IU/ml penicillin at
37°C in a
humidified atmosphere of 5% C02 in air.
Binding of [125I~glibenclamide (Amersham, UK) was performed in intact (3-cells
and in
so subcellular fraction derived therefrom. Isolated pancreatic islets from
ob/ob mice (250-300)
were homogenized in 300 ~1 buffer containing (in mM): HEPES 20, MgCl2 1, EGTA
1,
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EDTA 1, PMSF l, protease inhibitors (leupeptin, aprotinin, pepstatin,
antipain) 0.5 ~g/ml
in Eppendorf tubes using a motor pestle homogenizer. The homogenized material
was
centrifuged (800 x g for 10 min) and the resultant pellet was resuspended
again in 200 ~l of
homogenization buffer and centrifuged as above. Supernatants from both
centrifugations
were pooled for high-speed separation (100,000 x g for 30 min) of membranes
and cytosol.
Aliquots were incubated in the presence of 5 nM [1251]glibenclamide (2
~Ci/sample) with
or without x100 excess of cold glibenclamide. Samples were irradiated for 20
min by 312
nM UV light at room temperature. The reaction was terminated by placing the
samples on
ice and subjected to SDS-PAGE using standard procedures. Two-dimensional
io electrophoresis was performed in samples (200 mg protein) to which the
following was
added: urea powder (final concentration 6 M), isoelectric focusing sample
concentrate
(33.3% of 3.5-10 ampholine, 16.6% of 2-mercaptoethanol, 33.3% Triton-X 100,
16.6% of
10% SDS). Samples were incubated for 30 min at room temperature and loaded on
the gel.
After isoelectric focusing (900V, 14 h), the gels were taken out of the tubes,
soaked for 30
n min at room temperature in Laemmli buffer, and loaded on a 6.6-13.6%
gradient gel.
Following SDS-PAGE separation, gels were stained with Coomassie Brilliant Blue
R or
with silver (Silver Stain Plus kit, Bio-Rad, Richmond, CA). Localization of
the proteins
containing the radiolabeled [1251]glibenclamide was performed by
autoradiography.
zo Binding of [125I~glibenclamide to [3-cells in suspension isolated from
ob/ob mice, followed
by UV cross-linking and SDS-PAGE separation was performed to determine whether
this
compound interacts with cellular proteins other than the SUR1 unit of KATP-
channels. The
binding of [125I~glibenclamide to (3-cells performed under steady-state
conditions (30 min
at 4°C) was specific (Fig. 1 a), and sub-cellular fractionation
revealed that a great
zs proportion of such binding was localized to the plasma membrane, although
~30 % of the
binding remained associated with the cytosolic fraction. UV cross-linking did
not
significantly affect either the total binding or its distribution among the
cytosolic and
membrane fractions (Fig. la). This material was further analyzed by SDS-PAGE
separation
and autoradiography (Fig. 1b). Binding of [125I~glibenclamide to (3-cells was
associated
3o with a 145 kDa protein within the plasma membrane fraction. In the
cytosolic fraction,
glibenclamide associated with two proteins having a molecular mass of
approximately 47
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and 65 kDa, respectively (Fig. 1 b, left panel). After several washing steps
the binding of
~125I~glibenclamide remained in the supernatant of the cytosolic fraction for
the 65 and 47
kDa proteins (Fig. 1 b, middle panel) and in the pellet of the plasma membrane
fraction for
the 145 kDa protein (Fig. 1 b, right panel).
The 145 kDa protein was identified as the regulatory component (SUR-1 ) of the
K+-ATP
channel. The identities of the cytosolic proteins were investigated. The
cytosolic material
was separated using 2-dimensional gel electrophoresis (Fig. 1 c), and the
proteins were
excised for in-gel digestion and MALDI spectrum analysis. The analysis of the
47 kDa
to protein revealed that it is (3-tubulin. Peptides found in the MALDI
spectrum (19 of the
peptide masses) were applied to the MS-Fit peptide fingerprinting tool.
Peptide mass
fingerprinting was performed using the MS-Fit algorithm developed at the UCSF
Mass
Spectrometry Facility and available via http: //prospector. ucsf edu. For the
SwissProt
database search, the best hit was mouse tubulin [3-5 chain (16 matches out of
19), whereas
is in the NCBI non-redundant database, the best hit was mouse tubulin [3-3
chain (17 matches
out of 19). The database search for the ~65 kDa protein did not match any
known protein
sequence.
(b) Confocal microscopy analysis
Further analysis of sulfonylureas binding to (3-tubulin was performed using
confocal
microscopy in primary cultures of (3-cells obtained from ob/ob mice. Isolated
(3-cells in
culture were incubated in the presence or absence of different agonists for 3
min at 23°C.
The incubation time was terminated by fixation of the cells with 4%
formaldehyde in PBS
2, for 10 min at room temperature. After rinsing twice with PBS, cells were
incubated in
acetone at-20°C for 5 min and then quenched with PBS containing 1% BSA
for 30 min.
BODIPY~ FL glibenclamide was purchased from Molecular Probes, Inc., Catalog
Number
B-7439; http://www.probes.com). The green-fluorescent BODIPY~ FL fluorophore
has
excitation/emission maxima 503/512 nm. Staining with BODIPY~ FL glibenclamide,
3o primary anti (3-tubulin antibody (Amersham, UK) and secondary Texas Red~-
conjugated
antibody (Molecular Probes Inc.) was performed at room temperature for 1 h.
After rinsing
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with PBS the coverslips were mounted (SlowFade light, Molecular Probes,
Eugene, OR)
and examined using a confocal laser scanning microscope (Leica TCS NT, Leica
Lasertechnik GmbH, Heidelberg, Germany). Excitation wavelengths 488 nm and 568
nm
were used. The confocal microscope was equipped with an Ar/Kr laser, a double
dicroic
mirror for rhodamine/fluorescein and a 63x lens (Leica PL APO 63x/1.32-0.6
oil). Analysis
of the data was performed with the IMARIS and COLOCALISATION softwares
(Bitplane,
Zurich, Switzerland). Co-localization analysis of the data indicated an
interaction between
glibenclamide and (3-tubulin. Interestingly this interaction is mainly
localized to the interior
of the (3-cell and excluded the plasma membrane.
EXAMPLE 2: Sulfonylureas affects tubulin polymerization in vitro
To determine whether binding of glibenclamide also occurs in a cell-free
system, and
is whether it has any functional impact on the properties of tubulin, the rate
of tubulin
polymerization in vitro in the presence of glibenclamide was examined.
Polymerization of
tubulin leading to the formation of microtubules can be followed by observing
the increase
in optical density of a tubulin-containing solution at 340 nm. Tubulin
polymerization is
commonly initiated in vitro either by addition of a crucial buffer component
such as GTP
Zo or Mg2+ (Weisenberg, R.C. (1972) Science 177, 1104-1105) or, alternatively,
by an
increase in temperature.
Tubulin from bovine brain was purchased from Cytoskeleton Inc. in aliquots of
1 mg
lyophilized protein. Glibenclamide was purchased from Sigma and stocks
containing 4
2, mg/ml were prepared daily in dimethylsulfoxide (DMSO). Immediately prior to
the
microtubule growth experiments each milligram of tubulin was resuspended in
350 ~1 ice-
cold buffer solution (80 mM PIPES, 1 mM MgCl2, 1 mM EGTA and 10% glycerol at
pH
6.9) followed by addition of 1 mM ice-cold GTP (Sigma). The samples were mixed
and
kept on ice for 5 minutes. 170 ~l of this solution was subsequently
transferred to each of
so two ice-cold Eppendorff tubes containing 3.65 ~l of the glibenclamide stock
in DMSO or
just DMSO, respectively. Following mixing the solutions were transferred to
two cuvettes
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equilibrated at 15°C in a CARY 4E spectrophotometer equipped with a
thermostated
multicell holder. The samples were left in the cuvette holder at 15°C
for five minutes to
allow thermal equilibrium to be reached. The experiment was then started by
altering the
multicell holder set temperature to 35°C and the absorbance at 340 nm
was measured as a
s function of time. This was done simultaneously for both samples as the
multicell holder
changes between the two positions. Polymerization started as a result of the
increased
temperature and the effect of glibenclamide on the rate and extent of
polymerization could
be observed.
io The results are shown in Fig. 2. The rate of change in absorbance was
faster in the presence
of 0.17 mM glibenclamide and this is the case for three independent pairs of
tubulin
samples. It has previously been demonstrated that changes in OD are
proportional to the
concentration of microtubules, regardless of the length of these polymers
(Nagle, B.W. &
Bryan, J. (1976) Cold Spring Harbor Symposium on Cell Proliferation 3, 1213-
1232;
is MacNeal, R. K. & Purich, D. L. (1978) J. Biol. Chem. 253, 4683-4687).
Hence, it is clear
from Fig. 2a that although glibenclamide affects the rate of polymerization
there is not a
strong influence on the extent of polymerization.
2o EXAMPLE 3: Taxol inhibits, and nocodazole stimulates, sulfonylurea effects
on insulin
secretion
Taxol, a promotor of microtubule polymerization, and nocodazole, which induces
microtubule depolymerization, have earlier been shown to inhibit glucose-
stimulated
Zs insulin secretion from isolated rat islets of Langerhans (Howell, S.L. et
al. (1982)
Bioscience Reports 2, 795-801 ). Taxol and nodocazole were used to study the
functional
importance of (3-cell microtubules during the process of insulin secretion.
The basal medium used for isolation and for conducting the experiments was a
buffer
3o containing 12~ mM NaCI; 5.9 mM KCI; 1.3 mM CaCl2; 1.2 mM MgCl2; and 25 mM
HEPES (pH 7.4). BSA was added to the medium at the concentration of 1 mg/ml.
The
WO 01/13121 cA 02380983 2002-0l-23 pCT/sE00/01563
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dynamics of insulin release were studied by perifusing (3-cell aggregates
mixed with Bio-
Gel P4 polyacrylamide beads (BioRad, Richmond, CA), in a 0.5 ml column at
37°C
(Kanatsuna, T. et al. (1981) Diabetes 30, 231-234). The flow rate was 0.2
ml/min. Two-
min fractions were collected and insulin content was analyzed by
radioimmunoassay.
Cells were incubated with 5 ~M nocodazole for 30 min or 25 q,M taxol for 15
min prior to
starting perifusion of the cells. Taxol was present during the whole
experiment. The control
cells were incubated with their respective vehicle solutions. Protocols were
initiated at the
times indicated in Fig. 3 with either 1 ~M glibenclamide or 15 mM glucose. The
data
io represent the mean ~ SEM of four experiments.
Incubation with 5 ~,M nocodazole and 25 ~M Taxol did not affect the basal
level of insulin
secretion nor did they induce visible changes in the microtubule organization,
and the
architecture of the ~3-cells remained intact (Fig. 3a). In the presence of non-
stimulating (3
is mM) as well as stimulating (15 mM) concentrations of glucose, the presence
of 25 ~M of
Taxol prevented the increase in insulin secretion elicited by 1 ~,M
glibenclamide (Fig. 3b).
In contrast, under the same experimental conditions (3 and 15 mM glucose) but
in the
presence of 5 ~uM nocodazole, 1 ~,M glibenclamide induced a 5-fold increase in
insulin
secretion (at 3 mM glucose) and a one-fold increase at 15 mM glucose (Fig.
3c).
2o
These results suggest that a dynamic microtubule system is required for
sulfonylureas to
stimulate secretion of insulin. Moreover, they suggest that microtubule
dynamics are
probably essential for the traffic of insulin containing granules. When taxol,
which inhibits
tubulin turnover, is present the microtubules are "locked" in place and no
more transport
is can occur. Nocodazole, on the other hand, disrupts microtubules and as such
may promote
tubulin turnover/treadmilling. This may promote the transport of insulin
containing
granules to the point of secretion.
WO 01/13121 CA 02380983 2002-0l-23 PCT/SE00/01563
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EXAMPLE 4: Characterization of the 65 kDa polypeptide
Several strategies are employed to determine the identity of the ~65kDa
polypeptide that
binds sulfonylureas. Firstly, after sulfonylurea binding to pancreatic (3-cell
(intact or
isolated cytosol) the proteins are separated with 2-dimensional gel
electrophoresis. The
spot representing the 65 kDa polypeptide binding to glibenclamide is excised
and subjected
to MALDI analysis.
Additionally, after in vitro binding of sulfonylureas to (3-cell cytosol,
samples are separated
to by HPLC and fractions containing the radiolabeled protein separated on SDS-
PAGE, and
subject to gel staining and autoradiography. The radioactive band will be
excised from the
gel and sequenced.
