U.S. patent application number 10/219871 was filed with the patent office on 2003-02-06 for acc2-knockout mice and uses thereof.
This patent application is currently assigned to Research Development Foundation. Invention is credited to Abu-Elheiga, Lutfi, Matzuk, Martin M., Wakil, Salih J..
Application Number | 20030028912 10/219871 |
Document ID | / |
Family ID | 25012295 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030028912 |
Kind Code |
A1 |
Matzuk, Martin M. ; et
al. |
February 6, 2003 |
ACC2-knockout mice and uses thereof
Abstract
The present invention discloses transgenic mice with
inactivating mutations in the endogenous gene for the acetyl-CoA
carboxylase-2 isoform of acetyl-CoA carboxylase. Inactivation of
acetyl-CoA carboxylase-2 results in mice exhibiting a phenotype of
reduced malonyl-CoA levels in skeletal muscle and heart,
unrestricted fat oxidation, and reduced fat accumulation in the
liver and fat storage cells. As a result, the mice consume more
food but accumulate less fat and remain leaner than wild type mice
fed the same diet. These results demonstrate that inhibition of
ACC2 acetyl-CoA carboxylase could be used to regulate fat oxidation
and accumulation for purposes of weight control. The transgenic
mice of the instant invention provide a useful animal model to
identify such inhibitors and for studying the mechanisms of fat
metabolism and weight control.
Inventors: |
Matzuk, Martin M.;
(Pearland, TX) ; Abu-Elheiga, Lutfi; (Houston,
TX) ; Wakil, Salih J.; (Houston, TX) |
Correspondence
Address: |
Benjamin Aaron Adler
ADLER & ASSOCIATES
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
Research Development
Foundation
|
Family ID: |
25012295 |
Appl. No.: |
10/219871 |
Filed: |
August 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10219871 |
Aug 15, 2002 |
|
|
|
09749109 |
Dec 26, 2000 |
|
|
|
Current U.S.
Class: |
800/18 |
Current CPC
Class: |
G01N 33/5067 20130101;
G01N 33/5008 20130101; A01K 67/0275 20130101; A01K 2227/105
20130101; G01N 33/5088 20130101; G01N 33/502 20130101; C12N 9/93
20130101; G01N 33/5061 20130101; A01K 2217/05 20130101; C12N
15/8509 20130101; A01K 2267/0362 20130101 |
Class at
Publication: |
800/18 |
International
Class: |
A01K 067/027 |
Goverment Interests
[0001] This invention was produced in part using funds from the
Federal government under N.I.H. G.M. 19091. Accordingly, the
Federal government has certain rights in this invention.
Claims
What is claimed is:
1. A transgenic mouse, said mouse comprising a mutation in an
endogenous ACC2 gene for the acetyl-CoA carboxylase-2 isoform of
acetyl-CoA carboxylase, wherein said mutation inactivates said gene
and results in the lack of expression of a functional acetyl-CoA
carboxylase-2 isoform.
2. The mouse of claim 1 wherein one or more exons of said ACC2 gene
has been deleted.
3. The mouse of claim 2, wherein said exons have been replaced with
heterologous DNA sequences.
4. The mouse of claim 3, wherein said heterologous DNA sequences
comprise an HPRT expression cassette.
5. The mouse of claim 4, wherein an exon encoding a biotin binding
motif, of ACC2 is replaced with an HPRT expression cassette.
6. The mouse of claim 1, wherein said mouse exhibits a phenotype
comprising a metabolic reduction in malonyl-CoA production in
skeletal muscle and heart.
7. The mouse of claim 6, further comprising a phenotype of
unrestricted fat oxidation and reduced fat accumulation in the
liver and fat storage cells.
8. The mouse of claim 7, further comprising a phenotype of
consuming more calories than a wild type mouse yet accumulating
less fat than a wild type mouse.
9. A method of screening for an inhibitor of acetyl-CoA
carboxylase-2 isoform activity comprising the steps of:
administering potential inhibitors to wild type mice; and,
screening for mice which exhibit the phenotype of the transgenic
mouse of claim 8.
10. An acetyl-CoA carboxylase-2 inhibitor identified by the method
of claim 9.
11. A pharmaceutical composition comprising the acetyl-CoA
carboxylase-2 inhibitor of claim 10 and a pharmaceutically
acceptable carrier.
12. A method of inhibiting fat accumulation and promoting fatty
acid oxidation to promote weight loss or maintenance in said
individual comprising the step of administering a pharmaceutical
composition comprising an acetyl-CoA carboxylase-2 inhibitor of
claim 10 and a pharmaceutically acceptable carrier to, said
individual.
13. A method of obtaining a purified preparation of acetyl-CoA
carboxylase-1 protein which is free of acetyl-CoA carboxylase-2
comprising the step of: purifying said acetyl-CoA carboxylase-1
protein from tissues obtained from the transgenic mouse of claim
1.
14. A method of obtaining murine antibodies against acetyl-CoA
carboxylase-2 which are less crossreactive with acetyl-CoA
carboxylase-1 and other mouse proteins comprising the step of:
generating said antibodies in the transgenic mouse of claim 1.
15. A cell line derived from the transgenic mouse of claim 1.
16. The cell line of claim 15, wherein said cell line is derived
from cells selected from the group consisting of muscle cells,
heart cells, adipose cells, and liver cells.
17. A method of screening for agonists and antagonists of ACC2
comprising the steps of: administering a candidate compound to the
cell line of claim 15 and to cell lines derived from wild type
mice; and, monitoring said cell lines for alterations in cellular
activity, wherein a compound that specifically acts on ACC2 will
have alter cellular activity in wild type cells but will have no
effects on the cell line of claim 15.
18. The method of claim 17, wherein monitored cellular activities
are selected from the group consisting of mRNA expression, protein
expression, protein secretion, and lipid metabolism.
Description
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to the field of fat
metabolism and weight control. More specifically, the present
invention relates to the role of the ACC2 isoform of acetyl-CoA
carboxylase in regulating fatty acid accumulation and oxidation and
to transgenic mice deficient for the ACC2 isoform.
[0004] 2. Description of the Related Art
[0005] Acetyl-CoA carboxylase (ACC), a biotin-containing enzyme,
catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, an
intermediate metabolite that plays a pivotal role in the regulation
of fatty acid metabolism (1-3). It has been found that malonyl-CoA
is a negative regulator of carnitine palmitoyltransferase I (CPTI,
a component of the fatty-acid shuttle system (4,5) that is involved
in the mitochondrial oxidation of long-chain fatty acids. This
finding provides an important link between two opposed
pathways-fatty-acid synthesis and fatty-acid oxidation. Thus, it is
possible to interrelate fatty acid metabolism with carbohydrate
metabolism through the shared intermediate acetyl-CoA, the product
of pyruvate dehydrogenase. Consequently, the roles of malonyl-CoA
in energy metabolism in lipogenic (liver and adipose) and
non-lipogenic (heart and muscle) tissues has become the focus of
many studies (4-11).
[0006] In prokaryotes, acetyl-CoA carboxylase is composed of three
distinct proteins-the biotin carboxyl carrier protein, the biotin
carboxylase, and the transcarboxylase (12). In eukaryotes, however,
these activities are contained within a single multifunctional
protein that is encoded by a single gene. In animals, including
humans, there are two isoforms of acetyl-CoA carboxylase expressed
in most cells, ACC1 (M.sub.r.about.265,000) and ACC2
(M.sub.r.about.280,000), which are encoded by two separate genes
and display distinct tissue distribution (2-6, 13-17). Both ACC1
and ACC2 produce malonyl-CoA, which is the donor of the
"C.sub.2-units" for fatty acid synthesis and the regulator of the
carnitine palmitoyl-CoA shuttle system that is involved in the
mitochondrial oxidation of long-chain fatty acids (4, 5, 18).
Hence, acetyl-CoA carboxylase links fatty acid synthesis and fatty
acid oxidation and relates them with glucose utilization and energy
production because acetyl-CoA, the substrate of the carboxylases,
is the product of pyruvate dehydrogenase. This observation,
together with the finding that ACC1 is highly expressed in
lipogenic tissues such as liver and adipose and that ACC2 is
predominantly expressed in heart and skeletal muscle (3, 14, 17,
19), opened up a new vista in comparative studies of energy
metabolism in lipogenic and fatty acid-oxidizing tissues.
[0007] Diet, especially a fat-free one, induces the synthesis of
ACC's and increases their activities. Starvation or diabetes
mellitus represses the expression of the ACC genes and decreases
the activities of the enzymes. Earlier studies addressed the
overall activities of the carboxylases with specific
differentiation between ACC1 and Acc2. Studies on animal
carboxylases showed that these enzymes are under long-term control
at the transcriptional and translational levels and short-term
regulation by phosphorylation/dephosphorylation of targeted Ser
residues and by allosteric modifications induced by citrate of
palmitoyl CoA (16, 20-25). Several kinases have been found to
phosphorylate both carboxylases and to reduce their activities. In
response to dietary glucose, insulin activates the carboxylases
through their phosphorylation. Starvation and/or stress lead to
increased glycogen and epinephrin levels that inactivate the
carboxylases through phosphorylation (20-25). Experiments with rats
undergoing exercises showed that their malonyl CoA and ACC
activities in skeletal muscle decrease as a function of exercise
intensity thereby favoring fatty acid oxidation. These changes are
associated with an increase in AMP-kinase activity (25-28). The
AMP-activated protein kinase (AMPK) is activated by a high level of
AMP concurrent with a low level of ATP through mechanism involving
allosteric regulation and phosphorylation by protein kinase (AMP
kinase) in a cascade that is activated by exercise and cellular
stressors that deplete ATP (7-10). Through these mechanisms, when
metabolic fuel is low and ATP is needed, both ACC activities are
turned off by phosphorylation, resulting in low malonyl-CoA levels
that lead to increase synthesis of ATP through increased fatty acid
oxidation and decreased consumption of ATP for fatty acid
synthesis.
[0008] Recently, it was reported that the cDNA-derived amino acid
sequences of human ACC1 and ACC2 share 80% identity and that the
most significant difference between them is in the N-terminal
sequence of ACC2 (3, 13). The first 218 amino acids in the
N-terminus of ACC2 represents a unique peptide that includes, in
part, 114 of the extra 137 amino acid residues found in this
isoform (14). Polyclonal antibodies raised against the unique ACC2
N-terminal peptide reacted specifically with ACC2 proteins derived
from human, rat, and mouse tissues. These findings made it possible
to establish the subcellular localization of ACC1 and ACC2 (14) and
to later demonstrate that ACC2 is associated with the mitochondria
and that the hydrophobic N-terminus of the ACC2 protein plays an
important role in directing ACC2 to the mitochondria (6). ACC1, on
the other hand, is localized to the cytosol.
[0009] Although these findings and the distinct tissue distribution
of ACC1 and ACC2 suggest that ACC2 is involved in the regulation of
fatty acid oxidation and that ACC1 is involved in fatty acid
synthesis primarily in lipogenic tissues, they do not provide
direct evidence that the products of the genes ACC1 and ACC2 have
distinct roles.
[0010] The prior art is deficient in an understanding of the
separate roles of ACC1 and ACC2 have in the fatty acid metabolic
pathway. The prior art is also deficient in transgenic knockout
mice generated to lack ACC2 and methods of using these transgenic
mice. The present invention fulfills this long-standing need and
desire in the art.
SUMMARY OF THE INVENTION
[0011] Malonyl-CoA (Ma-CoA), generated by acetyl-CoA carboxylases
ACC1 and ACC2, is key metabolite in the regulation of fatty acid
(FA) metabolism. ACC1.sup.-/- mutant mice were embryonically
lethal, possibly due to lack of "C.sub.2-units" for the synthesis
of fatty acid needed for biomembrane synthesis. ACC2.sup.-/- mutant
mice bred normally and had normal life spans. ACC2.sup.-/- mice fed
normal diet did not accumulate fat in their livers as did the
wild-type mice and overnight fasting resulted in 5-fold increase in
ketone bodies production, indicating higher fatty acid oxidation.
ACC1 and fatty acid synthase activities and malonyl-CoA contents of
the livers of the ACC2.sup.-/- and ACC2.sup.+/+ mice were the same,
indicating that fatty acid synthesis is unperturbed yet the
malonyl-CoA was not available for the inhibition of the
mitochondrial fatty acid shuttle system, hence fatty acid oxidation
was relatively high.
[0012] Absence of ACC2 resulted in 30- and 10-fold lower
malonyl-CoA contents of muscles and heart, respectively. Fatty acid
oxidation in the ACC2.sup.-/- soleus muscles was 30% higher than
that of ACC2.sup.+/+ mice. Addition of insulin did not affect fatty
acid oxidation in the ACC2.sup.-/- soleus muscle, but, as expected,
it did reduce fatty acid oxidation by 50% in the wild-type soleus
muscle compared to that of the mutant. Isoproterenol, an analog of
glucagon, had little effect on fatty acid oxidation in the muscle
of the ACC2.sup.-/- mice but caused 50% increase in fatty acid
oxidation in the soleus muscle. The higher fatty acid oxidation in
the mutant mice resulted in 50% reduction of fat storage in the
adipose tissue compared to that of the wild-type mice. These
results are valuable to an understanding and control of fatty acid
metabolism and energy homeostasis in normal, diabetic, and obese
animals.
[0013] In one embodiment of the present invention, there is
provided a transgenic mouse having a mutation in an endogenous gene
for the ACC2 isoform of acetyl-CoA carboxylase that inactivates the
protein. The ACC2 gene may be mutated by deleting one or more exons
of the gene, which may be replaced by heterologous DNA sequences
such as an HPRT expression cassette. In a preferred embodiment, an
exon encoding a biotin-binding motif of ACC2 is replaced with an
HPRT expression cassette. These mice exhibit a phenotype consisting
of a reduction in malonyl-CoA levels in skeletal muscle and heart,
unrestricted fat oxidation, and reduced fat accumulation in the
liver and fat storage cells. The transgenic mice consume more food
than wild type mice but remain lean.
[0014] In yet another embodiment of the present invention, there is
provided a method of screening for an inhibitor of ACC2 isoform
activity consisting of the step of administering potential
inhibitors to wild type mice and screening for mice which exhibit
the same phenotype of the ACC2.sup.-/- transgenic mice.
[0015] In yet another embodiment of the present invention, there is
provided an ACC2 inhibitor identified by the above method. This
inhibitor may be incorporated into a pharmaceutical composition to
be administered to individuals for purposes of augmenting fatty
acid oxidation and inhibiting fat accumulation to promote weight
loss or maintenance.
[0016] In a further embodiment of the instant invention, a method
is described for obtaining a purified preparation of ACC1 protein
totally free of the ACC2 isoform by purifying ACC1 from the
Acc2.sup.-/- transgenic mice.
[0017] In another embodiment of the instant invention, a method is
provided for obtaining improved antibodies against ACC2 by
generating the antibodies in the Acc2.sup.-/- transgenic mice.
[0018] In yet another embodiment of the instant invention, cell
lines derived from the Acc2.sup.-/- transgenic mice are provided.
Cell lines derived from muscle, heart, adipose cells, and liver
cells are expected to be especially useful in bioassays and drug
targeting studies.
[0019] In yet another embodiment of the present invention, a method
of screening for agonists and antagonists of ACC2 is provided. This
method comprises the steps of administering candidate compounds
Acc2.sup.-/- cell lines and to cell lines derived from wild type
mice followed by experiments to detect alterations in cellular
activity. A compound that specifically acts on ACC2 will alter
cellular activity in wild type cells but have no effects on
Acc2.sup.-/- cells. Cellular activities that may be monitored
include mRNA expression, protein expression, protein secretion, and
lipid metabolism.
[0020] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the embodiments of the invention given for the purpose of
disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings form a
part of the specification. It is to be noted, however, that the
appended drawings illustrate embodiments of the invention and
therefore are not to be considered limiting in their scope.
[0022] FIG. 1A shows the strategy used to create the targeted
mutation. Of the two exons (dark boxes) that were identified in the
mouse genomic clone, the exon that contained the biotin-binding
motif (Met-Lys-Met) was replaced with an HPRT expression cassette
to generate the targeting construct. The 3' and 5' probes used to
identify the targeted events by Southern blot analysis are
indicated.
[0023] FIG. 1B shows a Southern blot analysis of the genomic DNAs
extracted from mouse tails. DNA's that were digested with BglI were
probed with the 5' probe; the DNAs digested with KpnI were probed
with the 3' probe. DNAs from the wild-type (+/+), heterozygous
(+/-), and ACC2-null (-/-) mice gave the expected fragment
sizes.
[0024] FIG. 1C shows a Northern blot of total RNA prepared from the
skeletal muscles of wild-type (+/+), heterozygous (+/-), and
ACC2-null (-/-) mice was probed with the .sup.32P-labeled 362-bp
cDNA fragment, which was used to screen the genomic library. The
probe detected a 10-kbp RNA band in the ACC2.sup.+/- and
ACC2.sup.+/+ RNAs but not in the ACC2.sup.-/- RNA. Hybridization of
the same filter (after stripping) with a mouse .beta.-actin cDNA
probe confirmed that equal amounts of RNA were loaded in the
gel.
[0025] FIG. 1D shows a confirmation of the absence of ACC2 protein
in the ACC2-null mice. Extracts (50 .mu.g each) from the livers,
skeletal muscles, and hearts of the mice were separated by SDS-PAGE
(6%). The proteins were transferred onto a nitrocellulose filter
and probed with avidin-peroxidase to detect biotin-containing
proteins. The locations of the two carboxylases--the 280-kDa ACC2
and the 265-kDa ACC1--are indicated.
[0026] FIG. 2 shows the levels of malonyl-CoA in the tissues of
wild-type (+/+) and ACC2 mutant (-/-) mice. The levels of
malonyl-CoA in the mouse tissue extracts were determined by the
incorporation of [.sup.3H]acetyl-CoA into palmitate in the presence
of NADPH and highly purified chicken fatty acid synthase (4, 29).
The [.sup.3H]palmitic acid synthesized was extracted with petroleum
ether and the radioactivity was measured. The mice were either fed
normal chow or were fasted for 48 hours before they were
sacrificed.
[0027] FIGS. 3A-3E show histological analyses of livers of 32 week
old male mice fed a standard diet. FIG. 3A shows livers of
wild-type (left) and Acc2.sup.-/- mutant mice (right after 24 hours
of starvation. Frozen sections of wild type and mutant livers were
stained with Oil Red-O to detect lipid droplets and counter-stained
with Mayer's hematoxylin. The liver sections of wild type mice
(FIG. 3B) show an abundance of red-stained lipid droplets compared
to the dramatic decrease in red-stained droplets in the
ACC2.sup.-/- mutant liver (FIG. 3C). Frozen sections of the same
livers were made from the same livers and stained for glycogen by
the periodic acid-Schiff method and counter-stained with
hematoxylin. The wild type livers (FIG. 3D) contain glycogen
(pink-stained) and unstained lipid vacuoles, whereas the mutant
livers (FIG. 3E) have little or no glycogen and few lipid
vacuoles.
[0028] FIG. 4 shows a summary of an experiment in which mice were
sacrificed by cervical dislocation, and the soleus muscles--two
from each hind lim--were resected from each mouse and were immersed
in 1.5 ml of Krebs-Henseleit buffer (pH 7.4) containing 4% fatty
acid-free bovine serum albumin, 10 mM glucose, and 0.3 mM
[9,10(n)-.sup.3H]palmitate (3 mCi/vial) [Ibrahimi, 1999 #423].
Where indicated, insulin (10 nM) or isoproterenol (3 mM) was added,
and the vials were incubated at 37.degree. C. under a humidified
O.sub.2/CO.sub.2 (95/5%) atmosphere for 30 min. At the end of
incubation period, the [.sup.3H].sub.2O was separated from the
labeled substrate and counted.
[0029] FIGS. 5A and 5B show food intake and growth (body weight) of
wild type and ACC2.sup.-/- mutant mice. Ten 3-week-old female
mice--5 wild type and 5 ACC2.sup.-/- mutants--were fed a standard
diet for 13 weeks. FIG. 5A: The weight of each mouse within each
group was measured weekly; the average and variance of the weights
are shown. FIG. 5B: Food intake was measured every week and was
expressed as cumulative food intake per mouse over the 13-week
period.
[0030] FIGS. 6A-6C show adipose tissue in Acc2.sup.-/- and wild
type mice. FIG. 6A shows a dorsal view of male littermates, aged 32
weeks, fed with a standard diet. Reduced white fat is observed
under the skin of the Acc2.sup.-/- mouse (-/-, 33.6 g weight)
compared with the wild type mouse (+/+, 34.2 g weight). FIG. 6B
shows abdominal view of the fat pads under the skin of Acc2.sup.-/-
(-/-) and wild type mice (+/+). FIG. 6C shows epididymal fat pads
isolated from the mutant (-/-, 0.75 g) and wild type (+/+, 1.4 g)
mice.
[0031] FIGS. 7A and 7B show the targeted mutation of the ACC1
locus. FIG. 7A shows the strategy used to create the targeted
mutation. The exon (dark box) that contains the biotin-binding
motif (Met-Lys-Met) was replaced with an HPRT expression cassette.
The 3' and 5' probes used for Southern blot analysis are
indicated.
[0032] FIG. 7B shows a typical pattern observed in genotyping by
Southern blot analyses of genomic DNA extracted from mouse tails.
The DNAs were digested with ShpI in duplicate. The blots were
probed with the 5' and 3' probes indicated in FIG. 7A. The presence
of only wild-type (+/+) and heterozygous (+/-) genotypes indicated
that no homozygous (-/-) mice were born.
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention provides a transgenic mouse having a
mutation in an endogenous ACC2 gene for the ACC2 isoform of
acetyl-CoA carboxylase, which results in the lack of expression of
a functional ACC2 isoform. This gene may be mutated by deleting one
or more exons of the ACC2 gene, which may be replaced by
heterologous DNA sequences such as an HPRT expression cassette.
Preferably, an exon encoding a biotin binding motif of ACC2 is
replaced with an HPRT expression cassette. The resulting mice
exhibit a phenotype consisting of a reduction in malonyl-CoA levels
in skeletal muscle and heart, unrestricted fat oxidation, and
reduced fat accumulation in the liver and fat storage cells. The
transgenic mice consume more food than wild type mice but
accumulate less fat.
[0034] The present invention also demonstrates a method of
screening for an inhibitor of ACC2 isoform activity consisting of
administering potential inhibitors to wild type mice and screening
for mice which exhibit the phenotype of the ACC2.sup.- transgenic
mice The present invention is also directed to an ACC2 inhibitor
identified by the above method. This inhibitor may be incorporated
into a pharmaceutical composition to be administered to individuals
for purposes of augmenting fatty acid oxidation and inhibiting fat
accumulation to promote weight loss or maintenance.
[0035] The instant invention also provides a purification method
for obtaining ACC1 protein that is free of the ACC2 isoform. This
is accomplished by purifying ACC1 from tissue obtained from the
Acc2.sup.-/- transgenic mice of the instant invention that lack the
ACC2 isoform.
[0036] The instant invention also provides for the preparation of
improved antibodies against ACC2 by generating the antibodies in
the Acc2.sup.-/- transgenic mice. Unlike wild type mice, these mice
are less immunologically tolerant of ACC2 since it is not present
during the development of immunological self-tolerance. As a
result, antibodies obtained from immunization of the Acc2.sup.-/-
transgenic mice with ACC2 are more directed to unique antigenic
domains of ACC2 than similar antibodies generated in wild type
mice.
[0037] The instant invention is further directed to cell lines
derived from the Acc2.sup.-/- transgenic mice. These cell lines are
useful in bioassays of ACC1 and ACC2 and in drug targeting studies.
Cell lines derived from the muscle, heart, adipose, and liver
tissues are especially useful in these studies.
[0038] The instant invention also includes a method of screening
for agonists and antagonists of ACC2. Candidate compounds are
administered to both Acc2.sup.-/- cell lines and wild type cell
lines. The cells are then monitored for alterations in cellular
function such a mRNA expression, protein expression, protein
secretions, and lipid metabolism. A compound that specifically acts
on ACC2 will have alter cellular activity in wild type cells but
will have no effect on the Acc2.sup.-/- cell line.
[0039] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
EXAMPLE 1
[0040] Generation ol ACC2.sup.- Transgenic Mice Based on the
homology between the human and mouse ACC2 genes (14), two
oligonucleotides from the biotin-binding region based on the cDNA
sequence of human ACC2 were designed: forward primer
(5'-CTGAATGATGGGGGGCTCCTGCTCT-3'; nucleotides 2551-2575) (SEQ ID
No. 1) and a reverse primer (5'-TTCAGCCGGGTGGACTTTAGCA- AGG-3';
nucleotides 2890-2913) (SEQ ID No. 2). These primers were used to
amplify cDNA from a QuickClone mouse heart cDNA pool (Clontech)
template.
[0041] The cDNA fragment obtained was sequenced and used to screen
a 129/SvEv mouse genomic library to isolate a 16-kbp .lambda.
genomic clone. By digesting the 16-kbp .lambda. genomic clone with
different restriction enzymes, a restriction map was established
and a gene targeting vector constructed that contained
positive-negative selection markers and lacked the exon that
contains the biotin-binding motif Met-Lys-Met (FIG. 1A). This
vector was used to generate murine 129SvEv ES cells with one mutant
copy of ACC2 gene (the mutant allele was termed Acc2.sup.tml LAE)
Two independent ES-cell clones were injected into mouse
blastocysts, which were then implanted into the uterine horns of
pseudopregnant females. Among the pups produced, eight high-level
chimeras were identified and crossbred with C57BL/6J females. Each
female gave birth to several agouti pups, indicating germ-line
transmission of the ES-cell genome. Southern blot analysis of
genomic DNA confirmed the presence of both the endogenous and the
disrupted alleles in the F1 heterozygotes. The heterozygous mice
were intercrossed, and their offspring were genotyped. Southern
blot analyses showed that the DNA hybridized with the 5' and 3'
probes shown in FIG. 1A and gave the signals expected from the
wild-type (+/+), heterozygous (+/-), and homozygous-null (-/-)
animals (FIG. 1B). After genotyping more than 300 mouse tails, it
was determined that 24% of the progeny were ACC2.sup.-/-, 22% were
ACC2.sup.+/+, and 54% were ACC2.sup.+/-; these results are
consistent with Mendelian inheritance. The ACC2.sup.-/- mutants
were viable, bred normally, and appeared to have a normal life
span.
EXAMPLE 2
[0042] ACC2 Expression in ACC2.sup.- Transgenic Mice
[0043] Northern blot analyses of total RNA of skeletal muscle
tissues resected from the wild-type, heterozygous, and
homozygous-null animals showed no detectable ACC2 mRNA in the
homozygous-null animals and, as expected, the level of ACC2 mRNA in
the heterozygous animals was half of that in the wild-type (FIG.
1C). Western blot analyses of heart, skeletal muscle, and liver
tissues from the ACC2.sup.-/- mutant mice using avidin peroxidase
to detect biotin-containing proteins showed no expression of ACC2
protein (FIG. 1D). The levels of ACC2 protein (280 kDa) were higher
than those of ACC1 protein (265 kDa) in the heart and skeletal
muscle tissues of the wild-type mice, whereas the ACC1 protein was
more predominant in their liver tissues. The absence of ACC2
protein in the ACC2.sup.-/- mutant mice was further confirmed by
confocal immunofluorescence microscopic analysis using
affinity-purified anti-ACC2-specific antibodies (6). Whereas the
hearts, skeletal muscles, and livers of the wild-type mice had
abundant expression of ACC2 antigen, there was no expression of
this protein in the ACC2.sup.-/- mutant mice (data not shown).
Thus, by all measurements, the Acc2 mutant allele is a null
allele.
EXAMPLE 3
[0044] Malonyl-CoA levels in ACC2.sup.- Transgenic Mice
[0045] Since the levels of malonyl-CoA in animal tissues are
attributed to the activities of both ACC1 and ACC2, the
consequences of the absence of ACC2 on the malonyl-CoA levels in
these tissues and whether ACC1 can compensate and, consequently,
raise the levels of malonyl-CoA in these tissues was determined. In
comparing the liver tissues of the wild-type and ACC2.sup.-/-
mutant mice, there were no significant differences in the
malonyl-CoA levels and overall ACC activities, suggesting that
almost all of the malonyl-CoA in the liver is contributed by ACC1
(FIG. 2).
[0046] On the other hand, in comparing the skeletal muscle and
heart tissues of the same two groups of mice, the levels of
malonyl-CoA to be about 30- and 10-fold lower, respectively, in
these tissues of the ACC2.sup.-/- mutant mice than in those of the
wild-type mice. This suggests that ACC2 is the main contributor of
malonyl-CoA in skeletal muscle and heart (FIG. 2). During fasting,
the levels of malonyl-CoA dropped comparably in the liver tissues
of both the wild-type and the ACC2.sup.-/- mutant mice, suggesting
that ACC1 is affected by dietary conditions (FIG. 2). The levels of
malonyl-CoA in the heart and muscle tissues of the fasted
ACC2.sup.-/- mutant mice were very low, suggesting that ACC1 in
these tissues is also affected by diet (FIG. 2). Since malonyl-CoA
in the muscle is generated primarily by ACC2 (3), starving the
wild-type mice reduced its levels by 70% from that in the muscles
of the well-fed mice, suggesting that the ACC2 activity in these
mice might be regulated by diet. ACC2 activity may be significantly
reduced by a decrease in the amount of ACC2 expressed or by
down-regulation of its activity or by both.
EXAMPLE 4
[0047] Fatty Acid Accumulation in ACC2.sup.- Transgenic Mice
[0048] Since the ACC reaction is the rate-determining step in fatty
acid synthesis (2) and because the levels of malonyl-CoA in the
livers of the wild-type and ACC2.sup.-/-0 mice were nearly the
same, fatty acid synthesis, which is determined by the level of
malonyl-CoA, would be the same in both mouse groups. Indeed, the
synthesis of palmitate measured by the incorporation of
.sup.14C-acetyl-CoA was the same for the livers of both the wild
type and mutant mice (data not shown). However, the fat content of
the two livers, specifically the triglyceride content, was
different. The livers of the wild-type mice stored more fat, as
evidence by their much lighter color compared to the livers of
mutant mice of equal age and sex that had been fed the same diet
(FIG. 3A). To confirm this supposition, liver tissues were stained
with Oil Red-O to detect lipids and quantitate their total lipid
and triglyceride content. The stained liver sections from adult
wild-type mice contained abundant lipid droplets (FIG. 3B), which
are primarily triglycerides, whereas those from the ACC2.sup.-/-
mutant mice contained significantly fewer lipid droplets (FIG.
3C).
EXAMPLE 5
[0049] Analysis of Total Lipids in the Liver of ACC2.sup.-
Transgenic Mice
[0050] Extracting the total lipids and analyzing them by thin-layer
chromatography further confirmed the significant reduction in the
accumulation of triglyceride in the liver. The total lipids of the
livers of the ACC2.sup.-/- mutant mice were 20% lower than those in
the livers of the wild-type mice, and the triglyceride content of
these lipids was 80% to 90% lower in the livers of the ACC2.sup.-/-
mutant mice than in those of the wild-type.
[0051] The ACC and fatty acid synthase (FAS) activities in liver
extracts of wild-type and ACC2.sup.-/- mutants were very similar
(data not shown). Thus, the difference in the liver lipid content
must have occurred because of uncontrolled mitochondrial fatty acid
oxidation in the livers of the ACC2.sup.-/-0 mutant mice, rather
than resulting from a reduction in fatty acid synthesis. Also,
since malonyl CoA is a negative regulator of the mitochondrial
carnitine palmitoyl-CoA shuttle system (5), it, absence in the
livers of the Acc2.sup.-/- mutant mice leads to increased fatty
acid translocation across the mitochondrial membrane, causing the
fatty acids to be available for as substrates for the
.beta.-oxidation that primarily occurs in the mitochondria.
Altogether, these results provide the strongest evidence thus far
that malonyl-CoA, which is synthesized by ACC2, does affect the
accumulation of lipids in the liver by controlling fatty acid
oxidation. Since ACC1-generated malonyl-CoA, abundant in the livers
of both groups of mice, apparently does not reduce the
.beta.-oxidation of fatty acids, it can be concluded that the
malonyl-CoA produced by ACC1 and ACC2 exist in two distinct
compartments of the cell--the cytosol and the mitochondria,
respectively and carry out distinct function in these
compartments.
EXAMPLE 6
[0052] Analysis of Glycogen in the liver of ACC2.sup.- Transgenic
Mice
[0053] Glycogen, the storage form of glucose in the liver and
muscles, plays an important role in energy homeostasis in animals
including humans. Its synthesis and degradation is closely related
to glucose metabolism. The enzymes involved in glycogen metabolism
are highly regulated by hormones such as insulin, glucagon, and
epinephrine. To test whether the Acc2.sup.- null mutation might
affect the level of liver glycogen, livers resected from wild-type
and Acc2.sup.-/- mutant mice were fixed in formaldehyde and stained
for glycogen by using the periodic acid-Schiff method.
[0054] In the nourished state, the livers of the wild-type mice
contained abundant amounts of glycogen (410.+-.10 .mu.mol/g wet
tissue). The synthesis and accumulation of glycogen in the livers
of the wild-type mice was expected when fed a normal diet since
high dietary carbohydrate leads to active glycolysis, generation of
ample energy, and the substrates required for the synthesis of
glycogen and fatty acids. Acc2 is highly active under these
metabolic conditions, and the malonyl-CoA that is generated
inhibits carnitine palmitoyl transferase I, which then leads to
decreased fatty acid oxidation. Nevertheless, the glycogen content
of the livers of the Acc2.sup.-/- mutant mice was 20% less than
that in the livers of the wild-type mice. The reason for this
difference has not been established, but it is hypothesized that
more glucose is utilized in the synthesis of fatty acids and their
subsequent oxidation in the Acc2.sup.-/- mutant mouse livers. In
the 24 hour fasted wild-type mouse liver shown in FIG. 3, glycogen
is clearly present (FIG. 3D), while in that of the Acc2.sup.-/-
mutant, there is little or no glycogen (FIG. 3E). In the 48
hour-fasted state, as expected, there was a significant drop in the
glycogen levels in the livers of the wild-type mice to 12.6.+-.1
.mu.mol/g wet tissue, and a further 12-fold drop was observed in
the liver glycogen content of the Acc2.sup.-/- mutant over that of
the wild-type mouse.
EXAMPLE 7
[0055] Fatty Acid Oxidation in ACC2.sup.- Transgenic Mice
[0056] To provide further evidence for the role of ACC21
synthesized malonyl-CoA as the regulator of fatty acid oxidation,
fatty acid oxidation was investigated in the mouse soleus muscle, a
type II muscle tissue responsive to hormonal regulation
(13-1510,11,28,29). As shown in FIG. 4, the oxidation of
[.sup.3H]palmitate was 30% higher in the isolated soleus muscles of
ACC2.sup.-/- mutant mice than in those of the ACC2.sup.+/+ mice.
Insulin is known to activate both ACC1 and ACC2 and, thereby, to
induce fatty acid synthesis and to reduce fatty acid oxidation,
respectively. Adding insulin to soleus muscles resected from
wild-type and from ACC2.sup.-/- mutant mice did not affect fatty
acid oxidation in the ACC2.sup.-/- mutant muscle cells (FIG. 4) but
did reduce palmitate oxidation by about 45% in the wild-type muscle
cells (FIG. 4). Based on these results, it can be concluded that
the insulin-mediated inhibition of .beta.-oxidation occurs through
the activation of ACC2, probably by (16,18,20,22-24)(7-10,
19-24).
[0057] The role of ACC2 in the regulation of mitochondrial
oxidation of fatty acids was further confirmed by using
isoproterenol, an analog of glucagon, which produces effects
opposite of those of insulin. Adding isoproterenol to wild-type
soleus muscle increased palmitate oxidation by 50% (FIG. 4),
raising it to nearly the same level as that found in the mutant
muscle cells. It is noteworthy that isoproterenol also further
increased fatty acid oxidation in the mutant soleus muscle cells
(FIG. 4). This additional increase may be due to factors
independent of malonyl-CoA (20). Altogether, these results confirm
for the first time that mitochondria-associated ACC2, and not
cytosolic ACC1, is responsible for the insulin-mediated activation
and isoproterenol (glucagon)-mediated inactivation that results,
respectively in decreased and increased fatty acid oxidation. Since
the mitochondrial CPTI activities of the soleus muscles from both
groups of mice were very similar (data not shown), the observed
effects of these hormones are solely due to their effect on
ACC2.
EXAMPLE 8
[0058] Analysis of Blood Glucose and Lipids in ACC2.sup.-0
Transgenic Mice
[0059] To determine the consequence of the ACC2-null mutation on
blood glucose and lipids, the serum levels of triglycerides,
cholesterol, and glucose were analyzed in wild-type and
ACC2.sup.-/- mutant mice fed a standard diet. The blood cholesterol
levels were similar in both groups of mice (92.8.+-.3.1 and
95.1.+-.7.4 mg per deciliter). The blood glucose levels were 20%
lower in mutant mice (176.6.+-.6.5 vs. 136.2.+-.54 mg per
deciliter), whereas the triglyceride levels were 30% higher in the
mutant mice fed a standard diet (35.1.+-.2.5 vs. 45.2.+-.59
mg/deciliter). The levels of the blood ketone bodies
(.gamma.-hydroxybutyrate) of both wild type and mutants maintained
on standard diet were very low--nearly undetectable. However,
fasting overnight (10-12 hr) increased the blood
.gamma.-hydroxybutyrate concentration of the ACC2.sup.-/- mice
5-fold over that of the wild type (2.5.+-.0.6 mM in mutant blood
vs. 0.7.+-.0.5 of the wild type), indicating that a significantly
higher degree of fatty acid oxidation had taken place in the mutant
mice over that of the wild type.
EXAMPLE 9
[0060] Feeding Experiments with ACC2.sup.- Transgenic Mice
[0061] Based on the results presented above, it appears that the
mitochondrial .beta.-oxidation of fatty acids occurred in the
ACC2.sup.-/- mutant mice in an unregulated yet sustained manner. To
understand the role of this type of fatty acid .beta.-oxidation and
its effect on food consumption and weight gain, feeding experiments
involving three groups of mice were carried out, each consisting of
one subgroup of 3- to 4-week-old female mice (5 wild-type and
ACC2.sup.-/- mutant mice) and a second subgroup of 3- to 4-week-old
male mice (5 wild-type and 5 ACC2.sup.-/- mutant mice), that were
fed a weighted standard diet ad liberatum (FIG. 5 represents a plot
of one of the groups). Food consumption (no spillage was noted) for
each group was measured every week for 15 to 20 weeks, and the
weight of each mouse was recorded weekly.
[0062] On the average, each ACC2.sup.-/- mutant mouse consumed
20-30% more food per week and maintained an average body weight of
21 g; in contrast, each wild-type mouse consumed 202 g of food per
week and maintained an average body weight of 23 g. The
ACC2.sup.-/- mutant mice are generally leaner, weighing about 10%
less than the wild-type mice throughout the feeding periods and
accumulating less fat in their adipose tissues as seen in FIG. 6.
For example, the epididymal fat pad tissue in an Acc2.sup.-/- male
weighed 0.7 g as compared to 1.5 g in a wild type male littermate.
Both aged 32 weeks and fed a normal diet (FIG. 6). These results
are noteworthy because they not only confirm the role of
ACC2synthesized malonyl-CoA in the regulation of mitochondrial
.beta.-oxidation, they also highlight the importance of
mitochondrial oxidation of fatty acids in the regulation of energy
homeostasis and in the regulation of fat storage in the adipose
tissue.
EXAMPLE 10
[0063] Generation of ACC1.sup.- Transgenic Mice
[0064] To demonstrate the importance of ACC1 in the de novo
synthesis of fatty acids, the same strategy was followed to
generate an ACC1-knockout mouse as done for ACC2. Like ACC2, the
ACC1 isoform is also highly conserved among animal species (3). A
forward primer (5'-GGATATCGCATCACAATTGGC-3') (SEQ ID No. 3 ) based
on the human ACC1 cDNA and a reverse primer
(CCTCGGAGTGCCGTGCTCTGGATC-3') (SEQ ID No. 4) that contained the
biotin-binding site was designed and used to amplify a 335-bp cDNA
probe using human cDNA as a template. A 129/SvEv mouse genomic
library was screened with the PCR fragment as described for ACC2,
and a 14-kbp clone was isolated, mapped with restriction enzymes,
and analyzed by Southern blotting (FIG. 7B). A correctly targeted
clone (FIG. 7A) was microinjected into C57BL/6J mouse blastocysts,
which were then implanted into the uterine horns of pseudopregnant
female mice. The male chimeras thus generated were bred with
C57BL/6J mates, and the ACC1 heterozygous offspring were
interbred.
[0065] After analyzing genomic DNA from more than 300 progenies by
Southern blotting using both the 5' and 3' probes, homozygous
ACCI-null mutant offspring were not obtained. The litter sizes were
less than average, being 6 or 7, and 35% of the progeny were
wild-type and 65% were heterozygous. These results demonstrate that
the ACC1 mutation is embryonically lethal.
[0066] To characterize this embryonic lethality, the mating of the
heterozygotes was timed and the resulting embryos were genotyped.
At gestation days E12.5 and E13.5, the viable embryos were 35%
wild-type and 65% heterozygous, indicating that the lethality had
occurred earlier. At gestation day E9.5, the remains of dead
embryos were recovered, and at gestation day E8.5, degenerating
embryos were recovered from inside the ectoplacental cone.
[0067] Discussion
[0068] Obesity is a major health factor that affects the body's
susceptibility to a variety of diseases such as heart attack,
stroke, and diabetes. Obesity is a measure of the fat deposited in
the adipose in response to food intake, fatty acid and triglyceride
synthesis, fatty acid oxidation, and energy consumption. Excess
food provides not only the timely energy needs of the body, but
promotes glycogen synthesis and storage in liver and muscle and
fatty acid and triglyceride synthesis and storage in the fat
tissues. Calorie restriction or starvation promotes glycogenolysis
that supplies glucose where needed and lipolysis that supplies
fatty acids for oxidation and energy production. Insulin and
glucagon are the hormones that coordinate these processes.
Malonyl-CoA is the key intermediate in fatty acid synthesis, has
recently assumed an additional role as a second messenger that
regulates energy levels (ATP) through fatty acid oxidation, which
in turn affects fatty acid synthesis and carbohydrate
metabolism.
[0069] The studies described above provide a definitive
characterization of the role of malonyl-CoA produced by ACC2 in the
regulation of fatty acid oxidation and energy metabolism.
Malonyl-CoA generated by ACC1 is the donor of the C.sub.2 units
required for fatty acid synthesis. Acetyl CoA, the substrate for
ACC1 and ACC2, is the product of pyruvate oxidation, hence studies
of the carboxylases interrelate three major metabolic
pathways-carbohydrate metabolism, fatty acid synthesis, and fatty
acid oxidation.
[0070] Studies on animal carboxylases, usually a mixture of ACC1
and ACC2, showed that these enzymes are under long-term control at
the transcriptional and translational levels and under short-term
regulation by phosphorylation/dephosphorylation of targeted Ser
residues and by allosteric modifications by citrate or
palmitoyl-CoA (4-6,11,16-24,29,31). Several kinases have been found
to phosphorylate both carboxylases and to reduce their activities.
Insulin activates the carboxylases through their dephosphorylation,
whereas glucagon and epinephrine inactivate them as a result of
their phosphorylation (7-9,20,22,24,25). The AMP-activated protein
kinase (AMPK), one of the most notable kinases, is activated by a
high level of AMP concurrent to a low level of ATP through
mechanisms involving allosteric regulation and phosphorylation by
protein kinase (AMPK kinase) in a cascade that is activated by
cellular stressors that deplete ATP (10). Through these mechanisms,
when metabolic fuel is low and ATP is needed, both the ACC
activities are turned off by phosphorylation, resulting in the low
malonyl-CoA levels that lead to increased synthesis of ATP through
increased fatty acid oxidation and decreased consumption of ATP for
fatty acid synthesis.
[0071] The differential expression of ACC1 and ACC2 in various
tissues-ACC1 is highly expressed in liver and adipose and ACC2 is
predominant in heart and muscle-and their cellular
localization--ACC1 in the cytosol and ACC2 on the mitochondrial
membrane--suggest that their functions are different though
interrelated. The cytosolic ACCI-generated malonyl-CoA is utilized
by the fatty acid synthase, which also is a cytosolic enzyme, for
the synthesis of fatty acids. The mitochondrial ACC2-generated
malonyl-CoA functions as a regulator of CPTI activity--CPTI being
the first enzyme that catalyzes the shuttling of long-chain fatty
acids into the mitochondria for .beta.-oxidation and energy
production. ACC2-generated malonyl-CoA, therefore, is a second
messenger that regulates ATP levels through fatty acid oxidation,
which, in turn, affects fatty acid synthesis and carbohydrate
metabolism.
[0072] The present studies of the ACC2 mutant mice strongly support
this conclusion. The levels of malonyl-CoA in the livers of the
mutant mice were similar to those in the livers of the wild-type
mice, indicating its synthesis by ACC1, the predominant carboxylase
in this tissue. In the livers of the wild-type mice, the
malonyl-CoA is used to synthesize fatty acids, which are then
converted into triglycerides that accumulate as lipid droplets
(FIG. 3A). In the livers of the ACC2.sup.-/- mutant mice, the
uncontrolled CPTI activity results in the oxidation of fatty acids
by the liver mitochondria or in the conversion of fatty acids into
lipids (very-low-density lipoproteins), which are then transported
through the bloodstream to the heart and muscles to overcome the
increased demand of these tissues for fatty acids consequential to
uninhibited CPTI activity and amplified fatty acid oxidation. These
conclusions were supported by the near absence of malonyl-CoA in
the heart and skeletal muscle tissues of the ACC2.sup.-/- mutant
mice, by the higher fatty acid-oxidation rate in the soleus muscles
of the ACC2.sup.-/- mutant mice, and by the occurrence of fatty
acid oxidation independent of insulin and isoproterenol, an analog
of glucagon (FIG. 5).
[0073] Finally, knocking out ACC2 in mice has demonstrated that the
lack of malonyl-CoA, the mitochondrial second messenger, produces
offspring that exhibit increased oxidation of fatty acids,
decreased accumulation of lipids, and decreased storage of glycogen
in the liver but are still morphologically normal, grow at an
expected rate, and breed normally (their longevity and aging are
being followed). All of the metabolic changes are expressed in food
consumption patterns and body weight--the ACC2.sup.-/- mutant mice
who were fed a standard diet typically consumed 20% more food than
did the wild-type mice yet eventually lost 10% of their body
weight. The implications of these results in human development and
disease remain to be explored.
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[0111] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0112] One skilled in the art will readily appreciate that the
present invention is well adapted to carry Out the objects and
obtain the ends and advantages mentioned, as well as those inherent
therein. It will be apparent to those skilled in the art that
various modifications and variations can be made in practicing the
present invention without departing from the spirit or scope of the
invention. Changes therein and other uses will occur to those
skilled in the art which are encompassed within the spirit of the
invention as defined by the scope of the claims.
Sequence CWU 1
1
4 1 25 DNA Artificial sequence Reverse oligonucleotide primer for
the PCR amplification of the biotin-binding region of ACC2 1
ctgaatgatg gggggctcct gctct 25 2 25 DNA Artificial sequence Reverse
oligonucleotide primer for the PCR amplification of the
biotin-binding region of ACC2 2 ttcagccggg tggactttag caagg 25 3 21
DNA Artificial sequence Forward oligonucleotide primer for the PCR
amplification of the biotin-binding region of ACC1 3 ggatatcgca
tcacaattgg c 21 4 24 DNA Artificial sequence Reverse
oligonucleotide primer for the PCR amplification of the
biotin-binding region of ACC1 4 cctcggagtg ccgtgctctg gatc 24
* * * * *