Note: Descriptions are shown in the official language in which they were submitted.
2186987
B&P File No. 3153-1 95 /LMK
Title: Inhibitors of UDP-GlcNAc:Galnl-3GalNAcaR 1~1-6 N-
acetylglucosaminyltransferase (core 2 GlcNAc-T) and Use of the Inhibitors to Prevent
or Treat Cardiomyopathy Associated with Diabetes
s
FIELD OF TIIE INVENTION
The invention relates to methods for preventing or treating cardiomyopathy
associated with diabetes mellitus and hyperglycemia by inhibiting UDP-GlcNAc:Gall31-
3GalNAc~R J31-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T); methods for
10 screening for substances that affect cardiomyopathy associated with diabetes mellitus and
hyperglycemia; and methods and ph~ ceutical compositions cont~inin~ the substances
for preventing or treating cardiomyopathy associated with diabetes mellitus and
hyperglycemia.
BACKGROUND OF TEIE INVENTION
Cardiovascular diseases are the major cause of morbidity and mortality in diabetic
patients, involving cardiac tissues as well as large vessels in the brain, heart, and lower
extremities (1). In the heart, the majority of the cardiac failure is probably due to
atherosclerotic processes in the ~lUI~y vessels, but multiple studies have documented that
a sizeable number of diabetic patients suffer from congestive heart failure without
20 significant coronary disease (2, 3). In addition, type I diabetic patients with ~ 5 yr of
disease have been reported to have abnormal cardiac function in the absence of significant
coronary vessel disease (4). These clinical finrlin~s are supported further by animal studies
documenting biochemical and functional changes in the cardiac tissue shortly after induction
of diabetes (5-8). From these results, it has been postulated that diabetes mellitus and its
25 metabolic sequelae can induce a specific form of cardiomyopathy (8, 9).
As with other chronic complications of diabetes, the cardio-vascular changes once
established are difficult to reverse, both in clinical and experimental settings (10-12). Most
cardiovascular abnormalities are metabolically induced with a great deal of interest directed
towards identifying alterations in gene ~x~ression induced by diabetes or hyperglycemia in
30 the vasculature. Since thickening of basement membrane is a classical finding in diabetes
microvasculature (10), many of the studies concerning glucose-reg~ ted genes have
2186987
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prim~rily focused on changes in the basement matrix components using cultured vascular
cells (13, 14).
SUMMARY OF TIIE INVENTION
The present illVt~llLOl:j have shown a direct association between UDP-GlcNAc:Gall31-
3GalNAcaR 131-6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T) and diabetic
cardiovascular disease. In particular, core 2 GlcNAc-T activity was increased by 82% in
diabetic hearts versus controls, while the enzymes GlcNAc-T1 and GlcNAc-TV responsible
for N-linked glycosylation were unchanged. The results indicate that core 2 GlcNAc-T is
specifically induced in the heart by diabetes or hyperglycemia.
Significantly, increased core 2 GlcNAc-T activity caused pathology in the heart of
diabetic experimental animal models which is similar to that observed in the heart of
diabetic patients after years with the condition. In particular, a transgenic mouse was made
with core 2 GlcNAc-T expression driven by a cardiac myosin promoter. At 4 months, a
marked hypertrophy of the left ventricle and general hypertrophy of the heart was observed.
The fin(lin~ by the present inventors indicate that inhibiting core 2 GlcNAc-T can
be useful in preventing or treating cardiomyopathy associated with diabetes and
hy~Jel~,lyc~ ia. It also permits the identification of substances which affect core 2 GlcNAc-
T and which may be used in the prevention and treatment of cardiomyopathy associated
with diabetes and hyperglycemia.
Therefore, broadly stated the present invention relates to a method of preventing or
treating cardiomy~pa~lly associated with diabetes and hypt;l~,lycelllia in a subject comprising
reducing core 2 GlcNAc-T activity. Levels of core 2 GlcNAc-T activity may be reduced by
~lmini~tering a substance which inhibits core 2 GlcNAc-T activity. Substances which
inhibit core 2 GlcNAc-T activity include known inhibitors of core 2 GlcNAc-T activity,
25 inhibitors identified using the methods described herein, and antisense sequences of a
nucleic acid sequence encoding core 2 GlcNAc-T activity.
The invention also relates to a method for screening for a substance that may be used
to prevent or treat cardiomyopathy associated with diabetes and hyperglycemia. In an
embodiment of the invention, a method of screening for a substance for use in preventing
30 or treating cardiomyopathy associated with diabetes and hyperglycemia is provided
comprising assaying for a substance that inhibits core 2 GlcNAc-T activity. A substance
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that inhibits core 2 GlcNAc-T activity may be identified by reacting core 2 GlcNAc-T with
an acceptor substrate and a sugar nucleotide donor in the presence of a substance suspected
of inhibiting core 2 GlcNAc-T, under conditions whereby the core 2 GlcNAc-T produces
a reaction product, detP.rmininE the amount of reaction product, and comparing the amount
5 of reaction product to an amount obtained for a control in the absence of the substance,
wherein lower amounts of reaction product with the substance indicate that the substance
inhibits core 2 GlcNAc-T.
Substances which inhibit core 2 GlcNAc-T may also be identified using the
methods of the invention by comparing the pattern and level of expression of core 2
10 GlcNAc-T in tissues and cells in the presence, and in the absence of the substance.
Substances which inhibit core 2 GlcNAc-T may also be assayed by treating a cell
which expresses core 2 GlcNAc-T with a substance which is suspected of inhibiting core
2 GlcNAc-T activity, and assaying for Gall31-3[GlcNAc~31-6]GalNAc~- associated with
the cell.
Substances which inhibit transcription or translation of the gene encoding core 2
GlcNAc-T may be identified by transfecting a cell with an ~xL ression vector comprising a
recombinant molecule cont~ining a nucleic acid sequence encoding core 2 GlcNAc-T, the
necessary elements for the transcription and/or translation of the nucleic acid sequence and
a reporter gene, in the presence of a substance suspected of inhibiting transcription or
20 translation of the gene encoding core 2 GlcNAc-T activity, and comparing the level of
~x~ression of core 2 GlcNAc-T or the expression of the protein encoded by the reporter
gene with a control cell transfected with the nucleic acid molecule in the absence of the
substance. The method can be used to identify transcription and translation inhibitors of the
gene encoding core 2 GlcNAc-T.
The substances identified using the method of the invention may be used to prevent
or treat cardiomyopathy associated with diabetes and hyperglycemia. Accordingly, the
substances may be formulated into ph~ ceutical compositions for adminstration toindividuals suffering from this condition.
Other objects, features and advantages of the present invention will become apparent
30 from the following detailed description. It should be understood, however, that the detailed
description and the specific examples while indicating preferred embodiments of the
invention are given by way of illustration only, since various changes and modifications
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within the spirit and scope of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF TIIE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figure 1 are blots of differential expressed cDNA fragments (A) and Northern blot
analysis (B and C) showing differential expression in control (C) and diabetic heart (D) of
the cDNA fragments identif1ed in Panel A;
Figure 2 shows the qll~ntit~tion of changes induced by diabetes in mRNA levels for
DH1 and clone 13 in heart and aorta;
Figure 3 shows the nucleotide and de~l1ced amino acid sequence of DH1 identifying
the sequence as core 2 GlcNAc-T;
Figure 4 is a Northern blot analysis for DH1 expression performed using 25 ~g total
cellular RNA isolated from age-matched non-obese diabetic (NOD) control mice (C) and
diabeticNOD mice(D);
Figure 5 is a Northern blot analysis for DH1 expression using 25 ,ug total cellular
RNA isolated from heart of control rat (C) and 2 week diabetic rat without insulin treatment
(D) or with insulin treatment for 1 week (D +I);
Figure 6 (A) is a Northern blot analysis using 20 ~4g total RNA isolated from aorta,
brain, heart, kidney, liver, solcus muscle, and lung of control (C) and 2 week diabetic (D)
20 rats, and the same blot reprobed with 36B4 cDNA as a control; and Panel (B) is a bar graph
showing the quanli~alive changes in DH1 levels induced by diabetes in aorta, brain, heart,
kidney, liver, lung, and skeletal muscle;
Figure 7 (A) is a blot of total cellular RNA from cultured cardiomyocytes (lanes 1-
3), fibroblasts (lane 4),and rat aortic smooth muscle cells (lane 5), probed with DH1; and
25 Panel (B) is a graph showing the qll~ntit~tic)n of changes induced by 22 mM glucose or 100
nM insulin for DH1 expression in cultured cardiomyocytes;
Figure 8 are graphs showing core GlcNAc-T, GlcNAc-T1, and GlcNAc-TV
activities in the heart of control (closed column) and two week diabetic rats; and
Figure 9 shows the nucleotide and amino acid sequence of human core 2 GlcNAc-T.
30 DETAILED DESCRIPTION OF THE INVENTION
As discussed above, the present invention relates to a method of preventing or
treating cardiomyopathy associated with diabetes and hyperglycemia comprising reducing
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core 2 GlcNAc-T activity. Levels of core 2 GlcNAc-T activity may be reduced by
administering a substance which inhibits core 2 GlcNAc-T activity, or inhibits transcription
or translation of the gene encoding core 2 GlcNAc-T.
Substances which inhibit core 2 GlcNAc-T activity include known inhibitors of core
5 2 GlcNAc-T. Examples of inhibitors of core 2 GlcNAc-T include an analog of the acceptor
substrate for core 2 GlcNAc-T such as GalBl-3GalNAcl~. Inhibitors of enzymes earlier on
in the Golgi olig s~c~h~ride processing pa~w~y may also be used to inhibit core 2 GlcNAc-
T derived product, for example UDP-Gal:GalNAcl~R~1-3 galactosyltransferase or UDP-
GalNAc:Ser/thr ~N-acetylg~l~ctosyltransferase.
Recombinant molecules cont~ining the nucleic acid sequence of core 2 GlcNAc-T
in antisense orientation may be used to inhibit core 2 GlcNAc-T activity. The nucleic acid
sequence shown in Figure 9 (see also GenBank Accession Nos. L41415, U41320, and
U19265 ), or parts thereof, may be inverted relative to their normal presentation for
transcription to produce antisense nucleic acid molecules. The antisense nucleic acid
15 molecules may be contructed using chemical synthesis and enzymatic ligation reactions
using procedures known in the art. The antisense nucleic acid molecules or a part thereof,
may be chemically synthP~i~Pd using naturally occurring nucleotides or variously modified
nucleotides dçcigned to increase the biological stability of the molecules, or to increase the
physical stability of the duplex formed with mRNA or the native gene e.g. phosphorothiate
20 de iv~ives and acridine substituted nucleotides. The antisense sequences may be produced
biologically using an expression vector introduced into cells in the form of a recombinant
plasmid, phagemid, or ~ttPml~tPd virus in which ~ntisP.n~e sequences are produced under the
control of a high efficiency regulatory region, the activity of which may be determined by
the cell type into which the vector is introduced.
The invention also provides methods for screening for a substance that may be used
to prevent or treat cardiomyopathy associated with diabetes and hyperglycemia. In an
embodiment of the invention, a method of screening for a substance for use in preventing
or treating cardiomyopathy associated with diabetes and hyperglycemia is provided
comprising assaying for a substance that inhibits core 2 GlcNAc-T activity. A substance
30 that inhibits core 2 GlcNAc-T activity may be identified by reacting core 2 GlcNAc-T with
an acceptor ~ub~ e and a sugar nucleotide donor in the presence of a substance suspected
of inhibiting core 2 GlcNAc-T, under conditions whereby the core 2 GlcNAc-T transfers
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the sugar nucleotide donor to the acceptor substrate to produce a reaction product,
detP.rmining the amount of reaction prQduct, and comparing the amount of reaction product
to an amount obtained for a control in the absence of the substance, wherein lower amounts
of reaction product with the substance indicate that the substance inhibits core 2 GlcNAc-T.
S The acceptor ~ul; sl-~le may be an oligos~cch~ride, a glycopeptide, or a glycoprolei~
having the following minim~l structure for the oligosaccharide portion Gal~ 3GalNAcl~-R
where R is any convenient group ~ linked covalently to the 1 position of the GalNAc
residue of the acceptor, for example p-nitrophenol, or octyl. The sugar nucleotide donor
is uridine diphospho-N-acetylglucosamine (UDP-GlcNAc) which can be labelled in the
10 GlcNAc portion with radioactive groups or other nonradioactive groups which can be used
for product detection. The concentration of the acceptor substrate and of the labelled UDP-
GlcNAc in the reaction may range from 0.01 mM to lOmM and the duration of the reaction
from 5 mimltes to 24 hours.
Conditions are selected so that the core 2 GlcNAc-T transfers the sugar nucleotide
15 donor to the acceptor ~ul)~ t~ to produce a reaction product. The substrate and sugar donor
are effective to interact with the core 2 GlcNAc-T within wide pH and temperature ranges,
for example from about 5 to 8 and from about 30 to 45~C, preferably from 37~C.
The core 2 GlcNAc-T may be obtained from commercial sources, or prepared by
expression of the gene encoding core 2 GlcNAc-T in host cells (for example, transfected
20 CHO cells). A substance that inhibits core 2 GlcNAc-T activity may also be identified by
treating a cell which expresses core 2 GlcNAc-T with a substance which is suspected of
affecting core 2 GlcNAc-T activity, and assaying for Gall31-3[GalNAcl31-6]GalNAcl~
associated with the cell. Gal~31-3[GalNAc~31-6]GalNA~ can be measured using a substance
that binds to the oligosaccharide product either alone or in association with an attached
25 glycoprotein. For example, cells expressing core 2 GlcNAc-T may be treated with a
substance suspected of inhibiting core 2 GlcNAc-T activity. An antibody specific for the
oli~s~crll~ride product can be added and the amount of antibody binding can be compared
to control cells which have not been treated with the substance and/or which do not express
core 2 GlcNAc-T. Antibodies specific for core 2 GlcNAc-T may be obtained from
30 commercial sources, for example lB 11 rat anti-mouse CD43 activation-associated isoform
monoclonal antibody supplied by Ph~rmin~en Inc.
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Substances which inhibit core 2 GlcNAc-T include substances which inhibit
transcription or translation of the gene encoding core 2 GlcNAc-T. Transcription inhibitors
may be identified by kansfecting a host cell with a recombinant molecule comprising a
nucleic acid sequence encoding core 2 GlcNAc-T, the necessary elements for the
5 transcription of the nucleic acid sequence, and a lepo-ler gene, in the presence of a
substance suspected of inhibiting transcription of the gene encoding core 2 GlcNAc-T, and
comparing the level of mRNA or expression of the protein encoded by the reporter gene
with a control cell transfected with the nucleic acid molecule in the absence of the
substance. Translation inhibitors may be identified by transfecting a host cell with a
10 recombinant molecule comprising a nucleic acid sequence encoding core 2 GlcNAc-T, the
necessary elements for the transcription and translation of the nucleic acid sequence, and
a l~o-ler gene, in the presence of a substance suspected of inhibiting translation of the gene
encoding core 2 GlcNAc-T, and comp~ring the level of expression of core 2 GlcNAc-T with
a control cell transfected with the nucleic acid molecule in the absence of the substance.
A recombinant molecule comprising a nucleic acid sequence encoding core 2
GlcNAc-T may be constructed having regard to the sequence of the core 2 GlcNAc-T gene
(see Figure 9) using chemical synthesis and enzymatic ligation reactions following
procedures known in the art.
Suitable transcription and translation elements may be derived from a variety of20 sources, including bacterial, fungal, viral, m~mm~ n, or insect genes. Selection of
app-up-iate transcription and translation elements is dependent on the host cell chosen, and
may be readily accomplished by one of ordinary skill in the art. Examples of such elements
include: a transcriptional promoter and enhancer, an RNA polymerase binding sequence,
a ribosomal binding sequence, including a translation initiation signal. Additionally,
25 depending on the host cell chosen and the vector employed, other genetic elements, such as
an origin of replication, additional DNA restriction sites, enhancers, and sequences
conferring inducibility of transcription may be incorporated into the expression vector. It
will also be appreciated that the necessary transcription and translation elements may be
supplied by the native gene and/or its fl~nkin~ sequences.
Examples of reporter genes are genes encoding a protein such as ~3-galactosidase(e.g. lac Z), chloramphenicol, acetyl-transferase, firefly luciferase, or an immunoglobulin
or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
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Transcription of the reporter gene is monitored by changes in the concentration of the
reporter protein such as 13-galactosidase, chloramphenicol ac~lylll~sferase, or firefly
luciferase. This makes it possible to visualize and assay for ~ression of recombinant
molecules to det~rmine the effect of a substance on expression of the core 2 GlcNAc-T
5 gene.
M~mm~ n cells suitable for carrying out the present invention include, for
example, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281),
CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), and 293 (ATCC No. 1573).
Suitable ~ ession vectors for directing expression in m~mm~ n cells generally include
10 a promoter. Common promoters include SV40, MMTV, metallothionein-l, adenovirus Ela,
CMV, immediate early, immunoglobulin heavy chain promoter and enhancer, and RSV-LTR.
Protocols for the transfection of m~mm~ n cells are well known in the art and
include calcium phosphate mediated electroporation, retroviral, and protoplast fusion-
15 mediated transfection (see Sambrook et al., Molecular Cloning A Laboratory Manual, 2ndedition, Cold Spring Harbor Laboratory Press, 1989).
The reagents suitable for applying the methods of the invention to identify
substances that may be used to prevent or treat cardiomyop~y associated with diabetes and
hyperglycemia may be packaged into convenient kits providing the necessary materials
20 packaged into suitable containers. The kits may also include suitable supports useful in
performing the methods of the invention.
Substances which inhibit core 2 GlcNAc-T activity may be incorporated into
ph~rmAceutical compositions. Therefore, the invention also relates to a pharmaceutical
composition comprising an inhibitor of core 2 GlcNAc-T activity and in particular a
25 substance identified using the methods described herein. The pharmaceutical compositions
of the invention contain the substance, alone or together with other active substances.
The substances identified using the method of the invention may be form~ ted into
ph~rm~r;elltical compositions for ~ Ll~lion to subjects in a biologically compatible form
suitable for arlmini~tration in vivo. By "biologically compatible form suitable for
30 a-lmini~tration in vivo" is meant a form of the substance to be arlmini~tered in which any
toxic effects are outweighed by the therapeutic effects. The substances may be arlmini~tered
to living org~ni~m~ including hllm~n~, and animals. Administration of a therapeutically
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g
active amount of the ph~rm~r.elltical compositions of the present invention is defined as an
amount ~.;liv~, at dosages and for periods of time necessary to achieve the desired result.
For example, a therapeutically active amount of a substance may vary according to factors
such as the disease state, age, sex, and weight of the individual. Dosage regima may be
5 adjusted to provide the o~ ulll therapeutic response. For example, several divided doses
may be ~rlmini~tered daily or the dose may be p-opol~ionally reduced as indicated by the
exigencies of the therapeutic situation.
The active substance may be ~lmini.~tered in a convenient manner such as by
injection (subcutaneous, intravenous, etc.), oral ~mini~tration, inhalation, transdermal
10 application, or rectal ~rlmini~tration. Depending on the route of ~lmini~tration, the active
substance may be coated in a m~trri~l to protect the compound from the action of enzymes,
acids and other natural conditions which may inactivate the compound.
The compositions described herein can be prepared by per se known methods for
the plep~lion of ph~rm~ceutically acceptable compositions which can be ~tlmini~tered to
15 subjects, such that an effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example,
in Remington's Ph~rm~ce~ltical Sciences (R~min~ton's Ph~rm~ceutical Sciences, Mack
Publishing Company, Easton, Pa., USA 1985). On this basis, the compositions include,
albeit not exclusively, solutions of the substances in association with one or more
20 ph~rm~ce~ltically acceptable vehicles or diluents, and contained in buffered solutions with
a suitable pH and iso-osmotic with the physiological fluids.
Recombinant molecules comprising an antisense sequence may be directly
introduced into cells or tissues in vivo using delivery vehicles such as r~ vil~l vectors,
adenoviral vectors and DNA virus vectors. They may also be introduced into cells in vivo
25 using physical techniques such as microinjection and electroporation or chemical methods
such as coprecipitation and incorporation of DNA into liposomes. Recombinant molecules
may also be delivered in the form of an aerosol or by lavage. Recombinant molecules
comprising an antisense sequence may also be applied extracellularly such as by direct
injection into cells.
Inhibitors of core 2 GlcNAc-T and ph~rm~ceutical compositions cont~ining the
inhibitors have pharmaceutical utility in the prevention and treatment of cardiomyopathy
associated with diabetes mellitus and hyperglycemia. The utility of the inhibitors of core
218698~
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2 GlcNAc-T and compositions of the invention may be confirmed in animal experimental
model systems.
The present invention also contemplates a transgenic non-human animal all of whose
germ cells and somatic cells contain a DNA construct introduced into the animal, or an
5 ancestor of the animal at an embryonic stage, the DNA construct when incorporated into the
germ line of the animal being adapted to develop cardiomyopathy similar to that associated
with diabetes mellitus and hypelglycemia. The transgenic animal of the invention is
therefore highly suited for investigating the molecular and cellular events involved in
cardiomyopathy associated with diabetes mellitus and hyperglycemia, and for in vivo
10 testing of the efficacy of drugs in the pl~v~lllion or treatment of cardiomyopathy associated
with diabetes mellitus and hyperglycemia.
In accordance with one embodiment of the invention, the transgenic non-human
animal contains a DNA construct comprising a gene encoding core 2 GlcNAc-T. In
accordance with a preferred embodiment of the invention, the transgenic non-human animal
15 contains a DNA construct comprising a gene encoding core 2 GlcNAc-T and a promoter
which stimlllAt~ ssion of the gene in the cardiovascular system. Suitable promoters
include the cardiac myosin promoter.
The Animal~ of the invention may be used to test substances for their efficacy in
preventing or treating cardiomyopathy associated with diabetes mellitus and
20 hyperglycemia. When an animal is treated with the substance to be tested and a reduced
incidence of cardiomyopalhy colllp~d to unllealed animals is observed, it is an indication
of the efficacy of the substance in the pl~v~lllion or treatment of cardiomyopathy associated
with diabetes mellitus and hyperglycemia.
The following non-limitin3~ examples are illustrative of the present invention:
Examples
Example 1
The following materials and methods were used in the Example:
Animals. Male Sprague-Dawley rats (Taconic Farms, Inc. German Town, NY) weighing180-200 grams were injected by the intraperitoneal route with STZ (80 mg/kg of body
30 weight). STZ was dissolved in 20mM cirate buffer (pH 4.5) immediately before use.
Cardiac tissue from spontaneous autoimmune-caused diabetic nonobese diabetic (NOD)
mice and their contral litt-~.rmAt~ were graciously provided by Dr. ~A~kA7:~1 Hattori of the
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11
Joslin Diabetes Cancer (Boston, MA) (19). Onset of diabetes was determined by the
detection of urinary glucose and confirmed by blood glucose levels. Insulin pellets
(T in~hin, Scarborough, Canada) were implanted subcutaneously 1 wk after STZ injection
in four STZ-diabetic rats for another week to normalize blood glucose level. The mean
5 plasma blood glucose level in insulin-treated STZ-diabetic rats was not significantly
different from control animals (4.8+0.5 vs 5.6+0.7 mM), while, the mean body weight in
the insulin-treated STZ-diabetic rats remained significantly less than control (270+17 vs
326+17 grams respectively, P ~ 0.01) but significantly greater than diabetic rats without
insulin treatment (210+33 grams), (P <0.01).
10 mRNA cl~ ial display. Rats were killed 2 wk after onset of diabetes. Cardiac ventricles
and thoracic aorta were dissected and washed with ice-cold PBS, immediatley frozen in
liquid N2, then crushed into frozen powder. Pieces of aorta from three rats were combined
into one sample. Total RNA was extracted using Ultraspec RNA isolation system (Biotecx
Laboratories, Houston, TX). mRNA differential display was performed as previously
15 reported (17, 18). Briefly, DNA-free RNA was obtained by treatment with DNase I
(GIBCO BRL, Grand Island, NY) in the presence of placental RNase inhibitor (GIBCO
BRL) for 30 min at 37~C. After phenol/chloroform extraction and ethanol precipitation, two
reverse transcriptions were performed for each RNA sample using 0.2,ug DNA-free total
RNA in lX reverse transcription buffer (PCR buffer) cont~inin~ 10 mM DTT, 20,uM each
20 of dGTP, dATP, dTTP, and dCTP, and 1,uM of either Tl2 NG or Tl2NC oligonucleotide
(Midland Certified Reagent Co., Midland, TX) where N is threefold degenerate for G, A,
and C. The solution was heated at 65~C for 5 min and cooled to 37~C, then superscript
reverse transcriptase (20 U) (GIBCO BRL) was added for 1 h. PCR was performed inreaction mixtures cont~ining 0.1 vol of reverse transcription reaction mixture, lX PCR
25 buffer, 2,uM each of dGTP, dATP, dTTP, and dCTP, 10 ,(LCi [~-3sS]-dATP, 1 ~M of the
respective Tl2NX oligonucleotide, 0.2 ,uM of 20 different specific arbitrary 10-mer
olignnll~leotides (OP-ERON Technologies Inc., Almeda, CA) and 10 U of AmpliTaq DNA
polymerase (Perkin-Elmer Cetus Corp. Norwalk, CT). The PCR reactions were initi~ted
at 95~C for 1 min, amplified 40 cycles at 94~C for 45 s, 40~C for 90 s, 72~C for 30 s, and
30 finished at 72~C for 15 min. DNA sequencing stop buffer (U.S. Biochemical, Inc.,
Cleveland, OH~ was added to samples which were heated at 80~C for 2 min before loading
on a 6% polyacrylamide sequencing gel (National Diagnostics, Atlanta, GA). After
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electrophorosis, the gels were exposed to XAR-5 film (Eastman Kodak Co., Rochester NY)
for 48 h. Bands evident under one glycemic condition and absent in the other were
identified and the PCR repeated to confirm the f1ndin$c.
Band recovery and Northern blot analysis. Bands reproducibly exhibiting significant
5 differences in expression were cut out and DNA was eluted by boiling in 10 mM Tris HCI
and lmM EDTA solution for 30 min. After ethanol precipitation, the DNA was reamplified
by PCR using applop-iate primers and conditions described above except for dNTP
concentrations of 20,uM and no radioisotope. Products were vi~u~li7ed on 2% agarose gels,
eluted, and used as probes for Northern blot analysis or subcloned. Total RNA (20-25 ~g)
10 was fractionated by d~n~lring 1% formaldehyde agarose gel electrophoresis and transferred
to Biotrans nylon membrane (ICN, Irvine, CA). 32P-labeled probes prepared by random
priming using a commercially available kit (Amersham Corp., Arlington Heights, IL) were
hybridized to W cross-linked blots in 0.1 M Pipes, 0.2 M NaPO4, 0.1 M NaCl, 1 mMEDTA, 5% SDS, and 60,ug/ml salmon sperm DNA at 65~C and washed in 0.5 x SSC, 5%
15 SDS at 65~C for over 1 h. mRNA expression was quantified using a phosphorImager and
standardized volume integration with the accompanying ImageQuant Analyzing Software
version 3.3 (Molecular Dynamics, Sunnyvale, CA) and loading differences were norm~li7ed
using 36B4 as standard cDNA probe (17, 18).
DNA sequencing. Samples showing significant changes by Northern blot analysis were
20 subcloned using the TA Cloning Kit (Invitrogen Corp., San Diego, CA). After the
subcloned inserts were checked by Northern blot analysis, DNA sequencing was performed
using commercially available Sequenase version 2.0 kit (U.S. Biochemical, Inc.). Gene
database searches were performed at the National Center for Biotechnology Information
(NCBI) using the BLAST network service.
25 Consfruction of the diabetic heart cDNA library. Poly(A) + RNA was isolated from the
total cellular RNA extracted from heart of diabetic rats using an oligo-dT cellulose column
(Ph~rrn~ri~ LKB Biotechnology Piscal~way, NJ) as previously described (17). cDNA was
pl~a~ed and ligated into the EcoRI sites of Lambda gt 10 (Stratagene, Inc., La Jolla, CA)
by standard methods (20). After p?lcL-~ging the DNA, Escherichia coli (C600) was infected
30 with the phage and plated on a P150 plate yielding about 5 x 104 independent plaque-
forming units. Plaques were lifted onto nitrocellulose (Schleicher & Schuell, Inc., Keene,
~186987
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NH) and cross-linked to the membrane by ultraviolet light. 20 P150 plates were screened,
which provided 1 x 106 plaque-forming units for screening.
Screening the cDNA library. A 214-bp cDNA (DHl) probe obtained from differentialdisplay was hybridized to the cDNA library by standard methods at 44~C then washed at
5 56~C (21). After screening 1 x 106 plaque-forming units, a positive cDNA insert was
isolated and subcloned into pBluescript (Stratagene, Inc.). For sequencing, the inserts were
restricted using BamH1 and EcoR1 and subcloned into pBluescript.
cDNA cloning of mouse UDP-GlcNAc:Gal,~1-3Gal/NAc~R J~1-6 N-
acetylglucosaminylfrcl"~rase (core 2 GlcNAc-T). App~ ately 2 x 105 colonies of a10 cDNA library preapred in pCDM8 anvitrogen Corp.) using poly A + RNA from D33W25,
a murine lymphoid tumor cell line (22), were screened by colony hybridization (23) to a
864-bp EcoRI-BamHl subcloned fragment of human core 2 GlcNAc-T isolated by PCR
(gift of Dr. A. Datti, Perugia, Italy) collespollding to amino acids 85-372 of the human
enzyme. Hybridization was performed overnight at 65~C in 500 mM sodium phosphate pH
15 7.2, 7% SDS, 1% BSA~ lmM EDTA. After rinsing, filters were washed at 65~C in 100 mM
sodium phosphate, 0.1% SDS. After three rounds of hybridization and purification, two
clones, designated C2-251 and C2-352, were isolated and gave specific and strongly
positive signals on Southern blots hybridized with the probe. The cDNA inserts were
subcloned as Xhol fragments into SalI cut pGEM5zf(+) (Promega Corp., Madison, WI) and
20 a series of exonuclease m-mung bean nuclease (GIBCO BRL) - nested deletions generated
from each end. DNA sequencing was performed using the Autoread sequencing kit and the
ALF DNA sequencer according to the m~n~lf~cturer's instructions (Pharmacia LKB
Biotechnology). Some sequences were also generated using internal primers. Data were
analyzed and edited using the UWGCG suite.
25 Transient expression of DHl in Cos 7 cells. A cDNA insert cn"l;1inillg the full open reading
frame of DH1 was isolated with Xhol and EcoRV and subcloned into pcDNAI/amp
(Invitrogen Corp.). The plasmid was purified by double CsC1 ultracentrifugation followed
by phenol/chloroform extraction (21), then DNA (0.5,ug) was transfected into conconfluent
Cos 7 cells cultured on P100 dishes using 20 ,ug of Lipofectin for 16 h at 37~C (GIBCO
30 BRL) (24). Cells were harvested 48 h later and used for measurement of core 2 GlcNAC
transferase activity.
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Preparation of cardiomyocytes. Cardiomyocytes were prepared by collagenase digestion
as described before (25). Briefly, hearts were excised and perfused through the aorta with
Krebs-Henseleit bicarbonate buffer cont~ining 5.5 mM glucose and 2.5 mM calcium. The
perfused medium was switched to the same buffer without calcium to stop contraction,
5 followed by a final perfusion with Krebs-Henseleit buffer cont~ining 50 ~M calcium, 0.1%
BSA, 312 U/ml hyaluronidase (Worthington, Freehold, NJ) and 0.1% collagenese
(Worthington). Ventricular tissue was dissociated by ~h~king in Krebs-Henseleit buffer
co~ g 50,uM calcium, 0.2% BSA, 312 U/ml hyaluronidase, and 0.1% collagenase. The
cells were allowed to settle under gravity and were washed twice in the Krebs-Henseleit
10 buffer co~ inil~g 100,uM calcium and 0.5% BSA before resuspension in minim~l essential
medium ~~ g Earle's salts, 26 mM sodium bicarbonate, 5mM creatine, 20mM Hepes,
100 U/ml penicillin G, 100 ,ug/ml ~ omycin, and 1.8 mM calcium. The cells wereseeded onto l~minin-coated dishes and m~int~ined in a 37~C humidified 0.5% air-5% CO2
incubator. All cells were allowed to equilibrate for 2 h then washed and refed with the same
15 media cont~inin~ 0.24 gram % BSA and cultured for 3 d with the media changed daily.
Some cells were cultured in the same media cont~ining either 22mM glucose or 10-7 M
insulin for 3 d with daily changes of media.
Measuremenf of core 2GlcNAc-Tactivity. Transfected Cos-7 cells were washed in PBS,
frozen, thawed, and lysed in 0.9% NaCl, 04.% Triton X-100 at 0~C. PBS-rinsed, fresh
20 frozen rat hearts were rinsed again in PBS and homogenized using a polytron in 0.9%
NaCl, 0.4% Triton X-100, 0.1 mM PMSF, 0.1% Trasylol at 0~C. The core 2 GlcNAc
rel~se reactions contained 50mM 2-(N-morpholino) ethanesulfonic acid (MFS) pH 7.0,
1 mM UDP-GlcNAc, 0.5 ,uCi UDP-6[3H]-N-acetylglucosamine (16,000 dpm/nmol, New
Fngl~n~ Nuclear, Boston, MA), 0.1 M GlcNAc, lmM of Gall31-3GalNAcK-pNp (Toronto
25 Research Chemicals, Toronto, Canada) as substrate, and 16,ul of cell lysate (8-12 mg/ml of
protein) for a final volume of 32 ,ul (26, 27). The GlcNAc-TV reactions were the same
except that Triton X-100 was added to a final concentration of 1%, and 1 mM of
GlcNAcl31-2ManKl-OManl31-O(CH2)8COOCH3 (Dr. O. Hindsgaul, University of Alberta,Ftlmnntnn, Canada, was used as acceptor (28). The GlcNAcTI reactions were the same as
30 GlcNAc-TV but with the addition of 10 mM MnCl2, and 1 mM ManKl-3 (ManKl-6)
Manl31-O(CH2)8 COOCH3 was used as acceptor (29). Reactions were incubated for either
1 or 2 h at 37~C, then diluted to 5 ml in H2O and applied to a Cl8 Sep-Pak (Millipore Corp.,
2186987
- 15-
Bedford, NA) in H2O, washed with 20 ml H2O. The products were eluted with 5 ml of
methanol and radioactivity was counted in a liquid scintillation ~-counter. Endogenous
enzymatic activity was measured in the absence of acceptor and subtracted from values
det~rrnined in the presence of added acceptor.
5 Sf~ analysis. Differences in signal intensity between controls and diabetic ~nim~l~
were expressed as pelcenlage of controls. Because percentages tends to deviate from
normal distribution, mean and standard error were calculated after transformation of data
to logarithmic values and data were expressed as mean (+SE range). Statistical analysis
(Student's t test) was performed using the logarithmic values.
10 RESULTS
Di~nlial display. The ~ression of mRNA species derived from the cardiac ventricles
of diabetic and control rats was ~IIIp~d using mRNA di~el~lltial display. Applux~ately
2,000, presumably different, mRNA species were screened in this study using 40
combinations of primer sets. As exemplified by Figure 1 (A), eight candidates appeared
15 differentially expressed when ventricular tissue from control and diabetic rats was
comp~d; five increased and three decreased their ~ression in the diabetic state. These
changes were confirmed by repetition at least twice using different prepal~ions of total
RNA.
Northern blot analysis. Signals from all the candidates were detectable by Northern blot
20 analysis using total RNA pl~al~ions. As shown in Figure 1 O, two of the eight candidate
species showed significant and reproducible changes in diabetic rats compared to controls.
Figure 2 d~m~n~ s that in rats diabetic for 2 wk, the level of DHl (5.0 kb) increased to
680% (580-790%, n = 8, P<0.001) of control in the heart but was not detectable in the
aorta. Furthermore, in rats with diabetes for just 1 wk, the mRNA level of DH1 in the heart
25 had already significantly increased to 410% of control levels. A significant increase of DH1
in the heart of diabetic rats was observed even after 4 wk of disease. The expression of
clone 13 mRNA increased to 350% (260-470%, n = 3, P<0.05) of control in the heart and
decreased to 43% (38-49%, n=3, P<0.05) of control in the aorta.
Sequence analysis of DHl and clone 13. The nucleotide sequences of cDNA fragments of
30 DH1 and clone 13 derived from differential display were det~.rmined. Both had fl~nking
primer sequences identical to those used in the differential display. Searching the national
gene databases (GenBank/EMBL) revealed that clone 13 had 99% identity to the Wistar rat
2186987
- 16-
mito~.honllri~l 16S ribosomal (30) while DHl, which was 214 bp in size, did not reveal any
homology to previously reported sequences.
Cloning offull-length DHl cDNA. To f~rilit~te identification, a cDNA library derived from
diabetic rat heart mRNA was screened using the 214-bp-cloned DHl PCR fragment as a
5 probe. Five overlapping recombinants were identified and the composite of the full cDNA
sequence was detP.rmined (~igure 3 (A)). It contained 5,010 bp inclusive of poly A tail and
corresponded to the size detected by the original Northern blot analysis . Open reading
frame analysis showed that the longest possible coding region which was from position 802
to 2085 and encoded 428 amino acids. The GXXATGC pattern was observed fl~nking the
10 region of the presumptive start codon (31) and a polyadenylation signal, AATAAA, was
found 15 bp up~lle~l from the polyA tail. Searches for homologous sequences in
Genbank/EMBL revealed that this cDNA shared 80% identity at the nucleotide level and
85% identity at the amino acid level with human core 2 (GlcNAc-T) (32). The mouse core
2 GlcNAc-T was also cloned and sequenced and it was found that DHl shared 92% identity
15 with the amino acid sequence of mouse core 2 GlcNAc-T (Figure 3 (B)). These findings
strongly suggested that DHl was rat core 2 GlcNAc-T.
C~ ation of DHl expression in the NOD mouse. To check that increased expression
of DHl was diabetes specific and not due to other effects of streptozotocin, DHl expression
in the hearts of spontaneous autoimmune-caused diabetic NOD mice was measured. As
20 shown in Figure 4, DHl hybridizing signals were detected by Northern blot analysis at 6.0,
4.6, and 1.9 kb from animals which had experienced 2-3 wk of hyperglycemia and diabetes.
The 4.6 and 6.0 kb bands in hearts from diabetic NOD mice increased to 560% of control
~nim~
Effects of insulin on DHl expression in diabetic rats. After 1 wk of diabetes, four rates
25 were treated with insulin for an ~drliti~n~l week. Blood glucose level was norm~li7ed from
24.7 to 4.8 mM (P<0.01). The Northern blot analysis shown in Figure 5, demonstrated that
cardiac expression of DHl in rats diabetic for 2 wk increased to 680% of control levels,
consisle~l with earlier data, whereas insulin treatment nnrm~li7~d the expression of the DHl
to 169% (134-214% P<0.001 vs 2-wk diabetic rats) of control levels.
30 Tissue d~sfribufion of DHl. Figure 6 (A) shows a representative Northern blot analysis of
DHl ~ ession using total cellular RNA isolated from tissues of control and diabetic rats.
Relative signal intensity was calculated using the 36B4 signal for norm~ tion and is
~186987
- 17-
shown in Figure 6 (B). In normal rats, DHl transcripts were relatively high in the brain,
kidney and liver and low in the heart, aorta, lungs, and skeletal (soleus) muscle. A
significant and cardiac specific increase (6.8-fold) in the expression of DH1 mRNA was
observed in diabetic animals.
5 The expression of DHl in cultured cells. Cultured cardiomyocytes were measured to
detP.rmine whether they could be the source of the increased expression of DH1. As shown
in Figure 7 (A,) DH1 hybridizing signals were detected by Northern blot analysis of cultured
cardiomyocytes at the sarne mobility as the mRNA from heart tissue (5.0 kb). However,
DH1 expression was not detectable in fibroblasts cultured from rat heart or rat aortic smooth
10 muscle cells even when using 25 ~g of total cellular RNA. Furthermore, in cardiomyocytes,
cultures elevating media glucose concentration from 5.5 to 22 mM increased the expression
of the DH1 by 78% (54-106%, P<0.05) while insulin (10-7M) decreased the expression by
53% (40-62%, P<0.05) of control levels as shown in Figure 7 (B).
Core 2 GlcNAc-Tactivity in cells transiently transfected with DHl . Although Cos 7 cells
15 lipofected with pcDNAVamp showed significant endogenous core 2 GlcNAc-T activity of
1.71+0.27 nmol/mg per h (n=3), cells transfected with the ~x~ression vector cont~ining a
full-length cDNA for DH1 in correct orientation had 3.85+1.6 nmol/mg per h (P<0.05,
n-3). The assay is specific for core 2 GlcNAc-T, as confirmed by analysis of the reaction
product Gal~31-3[GlcNAc~31-6]-GalNAc~-pNp by 1 H-NMR (Nuclear Magnetic Resonance)
20 and HPLC which had been reported in previous studies (28). Therefore, DH-1 encodes an
enzymatically active core-2 GlcNAc-T.
GlcNAc-T activity in heart. With the identification of DH1 being an enzyme involved in
me(li~ting the biosynthesis of O-linked sugar chains, the specificity of the diabetes effect
was tested by measuring the activities of core 2 GlcNAc-T (which branches maturing O-
25 linked sugar chains) and two other GlcNAc transferases (which are specific for branchingN-linked sugar chains) in the hearts of control and diabetic rats (Figure. 8). Core 2
GlcNAc-T activity increased significantly and specifically in diabetic hearts by 82% of
control (0.39+0.03 vs 0.71_0.10 nmoVh per mg protein n-3, P<0.05). In contrast, GlcNAc-
TI and GlcNAc-TV activities were not significantly different between control and diabetes
30 (GlcNAc-TI: 1.05_0.11 vs 0.79_0.09 nmol/h per mg protein, GlcNAc-TV:0.078_0.024 vs
0.077+0.023 nmol/h per mg protein). The changes thus seem restricted to O-glycosylation.
~186987
- 18-
While the present invention has been described with reference to what are presently
considered to be the preferred examples, it is to be understood that the invention is not
limited to the disclosed examples. To the contrary, the invention is intended to cover various
modifications and equivalent arrangements included within the spirit and scope of the
S appended claims.
All publications, patents and patent applications are herein incolllol~ted by reference
in their entirety to the same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be incorporated by reference in
its entirety.
2186987
- 19 -
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