U.S. patent application number 15/997383 was filed with the patent office on 2018-11-29 for milk fat globule epidermal growth factor 8 regulates fatty acid uptake.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Kamran Atabai, Amha Atakilit, Sayyed M. Amin Khalifeh-Soltani, Dean Sheppard.
Application Number | 20180340027 15/997383 |
Document ID | / |
Family ID | 52346723 |
Filed Date | 2018-11-29 |
United States Patent
Application |
20180340027 |
Kind Code |
A1 |
Atabai; Kamran ; et
al. |
November 29, 2018 |
MILK FAT GLOBULE EPIDERMAL GROWTH FACTOR 8 REGULATES FATTY ACID
UPTAKE
Abstract
Methods and compositions for regulating fatty acid uptake and/or
decreasing gastric motility in an animal are provided.
Inventors: |
Atabai; Kamran; (San
Francisco, CA) ; Khalifeh-Soltani; Sayyed M. Amin;
(San Francisco, CA) ; Sheppard; Dean; (Oakland,
CA) ; Atakilit; Amha; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
52346723 |
Appl. No.: |
15/997383 |
Filed: |
June 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14905065 |
Jan 14, 2016 |
10005838 |
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PCT/US2014/047061 |
Jul 17, 2014 |
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15997383 |
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61873134 |
Sep 3, 2013 |
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61856475 |
Jul 19, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23K 20/147 20160501;
A23L 2/52 20130101; C07K 16/2839 20130101; A23L 33/18 20160801;
C07K 16/2848 20130101; C07K 2317/76 20130101; A61K 38/1808
20130101; A23G 4/12 20130101; G01N 33/6893 20130101; G01N 2800/02
20130101; C07K 2317/24 20130101; A61P 1/00 20180101; A61K 2039/505
20130101; G01N 2800/044 20130101; A23L 2/66 20130101; A61K 38/1709
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A23L 33/18 20160101 A23L033/18; A23G 4/12 20060101
A23G004/12; A23L 2/52 20060101 A23L002/52; A23L 2/66 20060101
A23L002/66; A61K 38/17 20060101 A61K038/17; A23K 20/147 20160101
A23K020/147; A61K 38/18 20060101 A61K038/18; G01N 33/68 20060101
G01N033/68 |
Claims
1. A method of increasing fatty acid uptake and/or decreasing
gastric motility in an animal, the method comprising, administering
a polypeptide comprising (i) Milk Fat Globule Epidermal Growth
Factor 8 (Mfge8), or (ii) an integrin-binding portion of Mfge8, to
an animal in an amount sufficient to increase fatty acid uptake
and/or to decrease gastric motility in the animal.
2. The method of claim 1, wherein the Mfge8 is human Mfge8 (SEQ ID
NO:1) or is at least 80% identical to (i) SEQ ID NO:1 or (ii) an
integrin-binding portion of SEQ ID NO:1.
3. The method of claim 1, wherein the animal is human
4. The method of claim 3, wherein the animal is under two or one
years or under six, five, four, three, two, or one months old.
5. The method of claim 1, wherein the animal is a premature human
infant.
6. The method of claim 1, wherein the animal is diabetic or has
cystic fibrosis.
7. The method of claim 1, wherein the polypeptide is administered
intravenously, subcutaneously, intramuscularly, rectally, or
orally.
8. The method of claim 7, wherein the polypeptide is administered
as a component of a composition selected from the group consisting
of a powder, a tablet, a capsule, a lozenge, a chewing gum, a food
product, a supplemented beverage, or a medical food.
9. The method of claim 8, wherein the food product is infant
formula.
10. A composition comprising a polypeptide comprising (i) Milk Fat
Globule Epidermal Growth Factor 8 (Mfge8), or (ii) an
integrin-binding portion of Mfge8.
11. The composition of claim 10, wherein the Mfge8 is human Mfge8
(SEQ ID NO:1) or is at least 80% identical to (i) SEQ ID NO:1 or
(ii) an integrin-binding portion of SEQ ID NO:1.
12. The composition of claim 10, wherein the composition is
selected from the group consisting of a powder, a tablet, a
capsule, a lozenge, a chewing gum, a food product, a supplemented
beverage, or a medical food.
13. The composition of claim 10, further comprising a bovine milk
protein, a soy protein, betalactoglobulin, whey, soybean oil or
starch.
14. The composition of claim 12, wherein said supplemented beverage
is a member selected from the group consisting of an infant
formula, follow-on formula, toddler's beverage, milk, fruit juice,
and fruit-based drink.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Divisional of U.S. application
Ser. No. 14/905,065, filed Jan. 14, 2016, which is a National Stage
entry of International Application No. PCT/US2014/047061, filed
Jul. 17, 2014, which claims the benefit of U.S. Provisional
Application No. 61/873,134, filed Sep. 3, 2013 and U.S. Provisional
Application No. 61/856,475, filed Jul. 19, 2013, the disclosures of
which are each incorporated herein in their entireties for all
purposes.
REFERENCE TO A SEQUENCE LISTING, A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
[0002] This application includes a Sequence Listing as a text file
named "081906-1079740-215421US_ST25.txt", created on Mar. 14, 2018,
and containing 21,722 bytes. The material contained in this text
file is incorporated by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0003] Obesity is a central feature of the metabolic syndrome,
which leads to significant morbidity and mortality by increasing
the risk of diabetes and cardiovascular disease. The absorption of
dietary triglycerides with subsequent storage in adipose tissue is
a key step in the development of obesity (Berk, P. D., et al., J
Biol Chem 274, 28626-28631 (1999); Berk, P. D., et al., J Biol Chem
272, 8830-8835 (1997)). Under physiological conditions, cellular
uptake of fatty acids occurs primarily through protein-mediated
pathways consisting of a number of fatty acid transporters
expressed in tissue-specific patterns (Stump, D. D., Fan, X. &
Berk, P. D., J Lipid Res 42, 509-520 (2001); Anderson, C. M. &
Stahl, A., Mol Aspects Med 34, 516-528 (2013)). Translocation of
these transporters from the cytosol to the cell membrane is the
major mechanism through which the rate of fatty acid uptake can be
acutely regulated in response to dietary and metabolic cues (Stahl,
A. et al., Developmental cell 2, 477-488 (2002); Luiken, J. J., et
al., Am J Physiol Endocrinol Metab 282, E491-495 (2002); Luiken, J.
J., et al., Diabetes 52, 1627-1634 (2003)). Fatty acid transporter
translocation is regulated systemically by hormones and locally by
muscle contraction (Stahl, A. et al., Developmental cell 2, 477-488
(2002); Luiken, J. J., et al., Am J Physiol Endocrinol Metab 282,
E491-495 (2002); Luiken, J. J., et al., Diabetes 52, 1627-1634
(2003)).
[0004] Mfge8 is an integrin ligand (Hanayama, R., et al., Nature
417, 182-187 (2002)) that is highly expressed in the adipose tissue
of mice on a high-fat diet (HFD) (Aoki, N. et al., Endocrinology
148, 3850-3862 (2007)). Both the expression of Mfge8 and the
integrin receptors for Mfge8 are increased in the adipose tissue of
obese humans (Henegar, C. et al., Genome Biol 9, R14 (2008)).
Definitions
[0005] An integrin "antagonist" is any agent that competes with an
endogenous integrin ligand for available ligand binding sites on an
integrin.
[0006] A "therapeutic dose," "therapeutic amount," "therapeutically
effective amount," or "effective amount" of a molecule antagonist
is an amount of the molecule that prevents, alleviates, abates, or
reduces the severity of symptoms of a disease or condition to be
treated, e.g., a condition involving fatty acid uptake in a
patient.
[0007] As used herein, the terms "treat" and "prevent" are not
intended to be absolute terms. Treatment can refer to any delay in
onset, amelioration of symptoms, improvement in patient survival,
reduction of tissue damage, etc. Indeed, in some embodiments,
treatment according to the invention can result in reversal of the
disease. Similarly, prevention can refer to any delay in onset or,
depending on context, reduction in severity of symptoms. The effect
of treatment can be compared to an individual or pool of
individuals not receiving the treatment, or to the same patient,
e.g., before treatment.
[0008] The term "subject" is used broadly herein to refer to any
individual that is considered for treatment. Typically, the subject
is a human or some other mammal (e.g., an agricultural animal such
as a cattle, pigs, sheep, horses, or goats or a pet such as a dog
or a cat).
[0009] The term "antibody" refers to a polypeptide encoded by an
immunoglobulin gene or functional fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0010] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of
each chain defines a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
terms "variable heavy chain," "V.sub.H," or "VH" refer to the
variable region of an immunoglobulin heavy chain, including an Fv,
scFv, dsFv or Fab; while the terms "variable light chain,"
"V.sub.L" or "VL" refer to the variable region of an immunoglobulin
light chain, including of an Fv, scFv, dsFv or Fab.
[0011] Examples of antibody functional fragments include, but are
not limited to, complete antibody molecules, antibody fragments,
such as Fv, single chain Fv (scFv), complementarity determining
regions (CDRs), VL (light chain variable region), VH (heavy chain
variable region), Fab, F(ab)2' and any combination of those or any
other functional portion of an immunoglobulin peptide capable of
binding to target antigen (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul
ed., 4th ed. 2001). Various antibody fragments can be obtained by a
variety of methods, for example, digestion of an intact antibody
with an enzyme, such as pepsin; or de novo synthesis. Antibody
fragments are often synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., (1990) Nature 348:552). The term "antibody" also
includes bivalent or bispecific molecules, diabodies, triabodies,
and tetrabodies. Bivalent and bispecific molecules are described
in, e.g., Kostelny et al. (1992) J Immunol. 148:1547, Pack and
Pluckthun (1992) Biochemistry 31:1579, Hollinger et al. (1993),
PNAS. USA 90:6444, Gruber et al. (1994) J Immunol. 152:5368, Zhu et
al. (1997) Protein Sci. 6:781, Hu et al. (1996) Cancer Res.
56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et
al. (1995) Protein Eng. 8:301.
[0012] "Single chain Fv (scFv)" or "single chain antibodies" refers
to a protein wherein the V.sub.H and the V.sub.L regions of a scFv
antibody comprise a single chain which is folded to create an
antigen binding site similar to that found in two chain antibodies.
Methods of making scFv antibodies have been described in e.g., Ward
et al., Exp Hematol. (5):660-4 (1993); and Vaughan et al., Nat
Biotechnol. 14(3):309-14 (1996). Single chain Fv (scFv) antibodies
optionally include a peptide linker of no more than 50 amino acids,
generally no more than 40 amino acids, preferably no more than 30
amino acids, and more preferably no more than 20 amino acids in
length. In some embodiments, the peptide linker is a concatamer of
the sequence Gly-Gly-Gly-Gly-Ser, e.g., 2, 3, 4, 5, or 6 such
sequences. However, it is to be appreciated that some amino acid
substitutions within the linker can be made. For example, a valine
can be substituted for a glycine. Additional peptide linkers and
their use are well-known in the art. See, e.g., Huston et al.,
Proc. Nat'l Acad. Sci. USA 8:5879 (1988); Bird et al., Science
242:4236 (1988); Glockshuber et al., Biochemistry 29:1362 (1990);
U.S. Pat. No. 4,946,778, U.S. Pat. No. 5,132,405 and Stemmer et
al., Biotechniques 14:256-265 (1993).
[0013] As used herein, "chimeric antibody" refers to an
immunoglobulin molecule in which (a) the constant region, or a
portion thereof, is altered, replaced or exchanged so that the
antigen binding site (variable region) is linked to a constant
region of a different or altered class, effector function and/or
species, or an entirely different molecule which confers new
properties to the chimeric antibody, e.g., an enzyme, toxin,
hormone, growth factor, drug, etc.; or (b) the variable region, or
a portion thereof, is altered, replaced or exchanged with a
variable region, or portion thereof, having a different or altered
antigen specificity; or with corresponding sequences from another
species or from another antibody class or subclass.
[0014] As used herein, "humanized antibody" refers to an
immunoglobulin molecule in which CDRs from a donor antibody are
grafted onto human framework sequences. Humanized antibodies may
also comprise residues of donor origin in the framework sequences.
The humanized antibody can also comprise at least a portion of a
human immunoglobulin constant region. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. Humanization can be
performed using methods known in the art (e.g., Jones et al.,
Nature 321:522-525; 1986; Riechmann et al., Nature 332:323-327,
1988; Verhoeyen et al., Science 239:1534-1536, 1988); Presta, Curr.
Op. Struct. Biol. 2:593-596, 1992; U.S. Pat. No. 4,816,567),
including techniques such as "superhumanizing" antibodies (Tan et
al., J. Immunol. 169: 1119, 2002) and "resurfacing" (e.g., Staelens
et al., Mol. Immunol. 43: 1243, 2006; and Roguska et al., Proc.
Natl. Acad. Sci USA 91: 969, 1994).
[0015] As used herein, "V-region" refers to an antibody variable
region domain comprising the segments of Framework 1 (F1),
Complementarity Determining Region 1 (CDR1), F2, CDR2, and F3,
including CDR3 and F4, which segments are added to the V-segment as
a consequence of rearrangement of the heavy chain and light chain
V-region genes during B-cell differentiation. A "V-segment" as used
herein refers to the region of the V-region (heavy or light chain)
that is encoded by a V gene. The V-segment of the heavy chain
variable region encodes FR1-CDR1-FR2-CDR2 and FR3. For the purposes
of this invention, the V-segment of the light chain variable region
is defined as extending though FR3 up to CDR3.
[0016] As used herein, the term "J-segment" refers to a subsequence
of the variable region encoded comprising a C-terminal portion of a
CDR3 and the FR4. An endogenous J-segment is encoded by an
immunoglobulin J-gene.
[0017] As used herein, "complementarity-determining region (CDR)"
refers to one of the three hypervariable regions in each chain that
interrupt the four "framework" regions established by the light and
heavy chain variable regions. The CDRs are primarily responsible
for binding to an epitope of an antigen. The CDRs of each chain are
typically referred to as CDR1, CDR2, and CDR3, numbered
sequentially starting from the N-terminus, and are also typically
identified by the chain in which the particular CDR is located.
Thus, for example, a V.sub.H CDR3 is located in the variable domain
of the heavy chain of the antibody in which it is found, whereas a
V.sub.L CDR1 is the CDR1 from the variable domain of the light
chain of the antibody in which it is found.
[0018] The sequences of the framework regions of different light or
heavy chains are relatively conserved within a species. The
framework region of an antibody, that is the combined framework
regions of the constituent light and heavy chains, serves to
position and align the CDRs in three dimensional space. Thus, the
position of the CDRs within the V region is relatively conserved
between antibodies.
[0019] The amino acid sequences and positions of the CDRs and
framework regions can be determined using various well known
definitions in the art, e.g., Kabat, Chothia, international
ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al.,
supra; Chothia & Lesk, 1987, Canonical structures for the
hypervariable regions of immunoglobulins. J. Mol. Biol. 196,
901-917; Chothia C. et al., 1989, Conformations of immunoglobulin
hypervariable regions. Nature 342, 877-883; Chothia C. et al.,
1992, structural repertoire of the human VH segments J. Mol. Biol.
227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)).
Definitions of antigen combining sites are also described in the
following: Ruiz et al., IMGT, the international ImMunoGeneTics
database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc,
M.-P. IMGT, the international ImMunoGeneTics database. Nucleic
Acids Res. January 1; 29(1):207-9 (2001); MacCallum et al,
Antibody-antigen interactions: Contact analysis and binding site
topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et
al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al,
Methods Enzymol., 203, 121-153, (1991); Pedersen et al,
Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg M. J.
E. (ed.), Protein Structure Prediction. Oxford University Press,
Oxford, 141-172 1996).
[0020] The phrase "specifically (or significantly or selectively)
binds to" when referring to a given protein or peptide, refers to a
binding reaction which is determinative of the presence of the
protein in the presence of a heterogeneous population of proteins
and other biologics. Thus, under designated immunoassay conditions,
the specified antibodies or other antagonists bind to a particular
protein (e.g., for an .alpha.v.beta.5-specific antibody, an
.alpha.v.beta.5 integrin, .beta.5, or portions thereof, or for an
.alpha.v.beta.3-specific antibody, an .chi., .beta.3, or portions
thereof) and do not bind in a significant amount to other proteins
present in the sample. Specific binding to an antibody under such
conditions may require an antibody that is selected for its
specificity for a particular protein. For example, antibodies
raised against an .alpha.v.beta.5 integrins or a .beta.5
polypeptide can be further selected to obtain antibodies
specifically immunoreactive with that protein and not with other
proteins. In some embodiments, the specific antibody will also bind
to polymorphic variants of the protein, e.g., proteins at least
80%, 85%, 90%, 95% or 99% identical to a sequence of interest.
Generally, an .alpha.v.beta.5-specific antibody or an
.alpha.v.beta.3-specific antibody binds to the .beta. component of
the integrin as .alpha.v has a number of different binding
partners. However, as shown in the Example, an antibody specific
for .alpha.v is effective in blocking fat uptake because the
antibody targets both .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5.
[0021] "Specific" or "significant" binding are not intended to be
absolute terms. For example, if an antibody does not significantly
bind to a particular epitope, it binds with at least 5-fold,
8-fold, 10-fold, 20-fold, 50-fold, 80-fold, or 100-fold reduced
affinity as compared to the epitope against which the antibody was
raised. Binding affinity can be determined using techniques known
in the art, e.g., ELISAs. Affinity can be expressed as dissociation
constant (Kd or K.sub.D). A relatively higher Kd indicates lower
affinity. Thus, for example, the Kd of an .alpha.v.beta.5-specific
antibody for .alpha.v.beta.5 will typically be lower by a factor of
at least 5, 8, 10, 15, 20, 50, 100, 500, 1000, or more than the Kd
of the .alpha.v.beta.5-specific antibody with another protein. One
of skill will understand how to design controls to indicate
non-specific binding and compare relative binding levels.
[0022] A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays, Western blots, or
immunohistochemistry are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See, Harlow
and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, NY (1988) for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity. Typically, a specific or selective reaction will
be at least twice the background signal or noise and more typically
more than 10 to 100 times background.
[0023] An agent that "specifically competes" for binding reduces
the specific binding of an antibody to a polypeptide. A first
antibody is considered to competitively inhibit binding of a second
antibody, if binding of the second antibody to the antigen is
reduced by at least 30%, usually at least about 40%, 50%, 60%, 75%,
or at least about 90%, in the presence of the first antibody using
any of the competitive binding assays known in the art (see, e.g.,
Harlow and Lane, supra).
[0024] The term "equilibrium dissociation constant" or "affinity"
abbreviated (Kd or K.sub.D), refers to the dissociation rate
constant (k.sub.d, time.sup.-1) divided by the association rate
constant (k.sub.a, time.sup.-1 M.sup.-1). Equilibrium dissociation
constants can be measured using any known method in the art.
Antibodies with high affinity have a monovalent affinity less than
about 10 nM, and often less than about 500 pM or about 50 pM as
determined by surface plasmon resonance analysis performed at
37.degree. C. In some embodiments, the antibodies of the invention
have an affinity (as measured using surface plasmon resonance), of
less than 500 pM, typically less than about 100 pM, or even less
than 25 pM.
[0025] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. As used herein, the terms encompass amino acid
chains of any length, including full length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds. For example, the Mfge8 polypeptides described herein
can contain one of more non-naturally-occurring amino acid.
[0026] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0027] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0028] The terms "peptidomimetic" and "mimetic" refer to a
synthetic chemical compound that has substantially the same
structural and functional characteristics of Mfge8 polypeptides or
.alpha..sub.v.beta..sub.5 and/or .alpha..sub.v.beta..sub.5
antagonists. Peptide analogs are commonly used in the
pharmaceutical industry as non-peptide drugs with properties
analogous to those of the template peptide. These types of
non-peptide compound are termed "peptide mimetics" or
"peptidomimetics" (see, e.g., Fauchere, J. Adv. Drug Res. 15:29
(1986); Veber and Freidinger TINS p. 392 (1985); and Evans et al.
J. Med. Chem. 30:1229 (1987)). Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used
to produce an equivalent or enhanced therapeutic or prophylactic
effect. Generally, peptidomimetics are structurally similar to a
paradigm polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as a naturally occurring
.alpha.v.beta.5 ligand, but have one or more peptide linkages
optionally replaced by a linkage selected from the group consisting
of, e.g., --CH2NH--, --CH2S--, --CH2-CH2-, --CH.dbd.CH-- (cis and
trans), --COCH2-, --CH(OH)CH2-, and --CH2SO--. The mimetic can be
either entirely composed of synthetic, non-natural analogues of
amino acids, or, is a chimeric molecule of partly natural peptide
amino acids and partly non-natural analogs of amino acids. The
mimetic can also incorporate any amount of natural amino acid
conservative substitutions as long as such substitutions also do
not substantially alter the mimetic's structure and/or
activity.
[0029] As used herein, the terms "nucleic acid" and
"polynucleotide" are used interchangeably. Use of the term
"polynucleotide" includes oligonucleotides (i.e., short
polynucleotides). This term also refers to deoxyribonucleotides,
ribonucleotides, and naturally occurring variants, and can also
refer to synthetic and/or non-naturally occurring nucleic acids
(i.e., comprising nucleic acid analogues or modified backbone
residues or linkages), such as, for example and without limitation,
phosphorothioates, phosphoramidates, methyl phosphonates,
chiral-methyl phosphonates, 2-O-methyl ribonucleotides,
peptide-nucleic acids (PNAs), and the like. Unless otherwise
indicated, a particular nucleic acid sequence also implicitly
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences as well
as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues (see,
e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et
al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al. (1992);
Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
[0030] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid that encodes a polypeptide is implicit in each described
sequence.
[0031] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0032] The following eight groups each contain amino acids that are
conservative substitutions for one another:
1) Alanine (A), Glycine (G);
[0033] 2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
[0034] 8) Cysteine (C), Methionine (M) (see, e.g., Creighton,
Proteins (1984)).
[0035] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (e.g., a polypeptide of the invention), which
does not comprise additions or deletions, for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0036] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same sequences. Two
sequences are "substantially identical" if two sequences have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%,
90%, or 95% identity over a specified region, or, when not
specified, over the entire sequence), when compared and aligned for
maximum correspondence over a comparison window, or designated
region as measured using one of the following sequence comparison
algorithms or by manual alignment and visual inspection. The
invention provides polypeptides that are substantially identical to
the polypeptides exemplified herein (e.g., any of SEQ ID NO: 1, 2,
3, 4, 5, or 6). Optionally, the identity exists over a region that
is at least about 50 nucleotides in length, or more preferably over
a region that is 100 to 500 or 1000 or more nucleotides in length,
or the entire length of the reference sequence.
[0037] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0038] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0039] Two examples of algorithms that are suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al. (1977)
Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol.
Biol. 215:403-410, respectively. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information. This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0040] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
BRIEF SUMMARY OF THE INVENTION
[0041] Methods of reducing fatty acid uptake in an animal are
provided. In some embodiments, the method comprises administering
an antagonist of integrin .alpha.v.beta.3 or .alpha.v.beta.5 to an
animal in an amount sufficient to reduce fatty acid uptake in the
animal, thereby reducing fatty acid uptake in the animal. In some
embodiments, the animal is a human.
[0042] In some embodiments, the antagonist is an antibody In some
embodiments, the antibody specifically binds to .alpha.v.beta.3. In
some embodiments, the antibody specifically binds to
.alpha.v.beta.5 In some embodiments, the antibody is a humanized or
chimeric antibody. In some embodiments, the antibody binds the same
epitope as ALULA (the antibody produced by the hybridoma deposited
under ATCC Deposit No. PTA-5817).
[0043] In some embodiments, the animal is obese.
[0044] In some embodiments, the animal has reduced insulin
sensitivity.
[0045] In some embodiments, the animal is insulin resistant.
[0046] In some embodiments, the antagonist is administered
intravenously, intraperitoneally, subcutaneously, intramuscularly,
rectally, or orally.
[0047] Also provided is a method of increasing fatty acid uptake
and/or gastric motility in an animal. In some embodiments, the
method comprises administering a polypeptide comprising (i) Milk
Fat Globule Epidermal Growth Factor 8 (Mfge8), or (ii) an
integrin-binding portion of Mfge8, to an animal in an amount
sufficient to increase fatty acid uptake and/or to gastric motility
in the animal.
[0048] In some embodiments, the Mfge8 is human Mfge 8 (SEQ ID NO:1)
or is at least 80% identical to (i) SEQ ID NO:1 or (ii) an
integrin-binding portion of SEQ ID NO:1.
[0049] In some embodiments, the animal is human. In some
embodiments, the animal (e.g., human) is under two or one years or
under six, five, four, three, two, or one months old. In some
embodiments, the animal is a premature human infant. In some
embodiments, the animal is diabetic or has cystic fibrosis.
[0050] In some embodiments, the polypeptide is administered
intravenously, intraperitoneally, subcutaneously, intramuscularly,
rectally, or orally. In some embodiments, the polypeptide is
administered as a component of a composition selected from the
group consisting of a powder, a tablet, a capsule, a lozenge, a
chewing gum, a food product, a supplemented beverage, or a medical
food. In some embodiments, the food product is infant formula.
[0051] Also provided is a composition comprising a polypeptide
comprising (i) Milk Fat Globule Epidermal Growth Factor 8 (Mfge8),
or (ii) an integrin-binding portion of Mfge8. In some embodiments,
the Mfge8 is human Mfge 8 (SEQ ID NO:1) or is at least 80%
identical to (i) SEQ ID NO:1 or (ii) an integrin-binding portion of
SEQ ID NO:1.
[0052] In some embodiments, the composition is selected from the
group consisting of a powder, a tablet, a capsule, a lozenge, a
chewing gum, a food product, a supplemented beverage, or a medical
food. In some embodiments, the composition further comprises a
bovine milk protein, a soy protein, betalactoglobulin, whey,
soybean oil or starch. In some embodiments, said supplemented
beverage is a member selected from the group consisting of an
infant formula, follow-on formula, toddler's beverage, milk, fruit
juice, and fruit-based drink.
BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIGS. 1A-1I. Mfge8 mediates fatty acid uptake in adipocytes.
(A) Fatty acid uptake in undifferentiated 3T3-L1 fibroblasts and
differentiated 3T3-L1 adipocytes treated with the rMfge8 or RGE
construct. N=4. (B) 3T3-L1 adipocyte triglyceride content after
treatment with the rMfge8 or RGE construct (10 .mu.g/ml). N=3.
(C-F) Fatty acid uptake in Mfge8-/- primary adipocytes (C, N=7-9),
differentiated primary Mfge8-/- adipocyte progenitors cells (D,
N=3), and 3T3-L1 adipocytes (F, N=4) after incubation with mutated
Mfge8 constructs (E). (G,H) Effect of integrin blocking antibodies
on fatty acid uptake in Mfge8-/- adipocytes treated with rMfge8 (G,
N=3-4) and in 3T3-L1 adipocytes (H, N=5). (I) Fatty acid uptake in
.beta.5-/- and .beta.3-/- primary adipocytes with and without the
addition of integrin blocking antibodies. N=4. *P<0.01,
**P<0.001, ***P<0.0001. Data are expressed as mean.+-.s.e.m.
Each replicate represents an independent experiment.
[0054] FIGS. 2A-2D. Mfge8 mediates fatty acid uptake in hepatocytes
and cardiac myocytes. (A) Fatty acid uptake in primary Mfge8+/+ and
Mfge8-/- hepatocytes, and Mfge8-/- hepatocytes treated with rMfge8
or RGE. N=6. (B) Fatty acid uptake in primary Mfge8+/+ and Mfge8-/-
cardiac myocytes and Mfge8-/- cardiac myocytes treated with rMfge8
or RGE. N=6. (C) The effect of mutated Mfge8 constructs on fatty
acid uptake by HepG2 cells. N=4. (D) The effect of integrin
blocking antibodies (20 .mu.g/mL) on fatty acid uptake in HepG2
cells treated with rMfge8. *P<0.01, **P<0.001,
***P<0.0001. Data are expressed as mean.+-.s.e.m. Each replicate
represents an independent experiment.
[0055] FIGS. 3A-3K. Mfge8 mediates fatty acid uptake in the
intestinal tract. (A) Triglyceride content of the small intestine
in mice fasted for 8 hours. N=5. (B) Fatty acid uptake in primary
Mfge8+/+ and Mfge8-/- enterocytes, and Mfge8-/- enterocytes treated
with rMfge8 or RGE. N=8. (C) Serum triglycerides after oral gavage
of Mfge8+/+ and Mfge8-/- mice with olive oil and Mfge8-/- mice with
olive oil mixed with rMfge8 or RGE construct. N=3-5, *P<0.01,
***P<0.0001 when comparing Mfge8-/- with Mfge8+/+ mice,
P<0.01, P<0.05 when comparing Mfge8-/- mice with Mfge8-/-
treated with rMfge8, #P<0.01 when comparing Mfge8+/+ mice with
Mfge8-/- treated with rMfge8. (D) Liver triglyceride levels 8 hours
after olive oil gavage as described for FIG. 3C. N=3-5. (E) Serum
free fatty acid levels in Mfge8+/+ and Mfge8-/- mice after olive
oil gavage, N=4-5. (F) Effect of rMfge8 or RGE construct on serum
triglycerides after olive oil gavage in Mfge8+/+ mice. N=4-5. (G)
Fecal fatty acid levels in Mfge8+/+ and Mfge8-/- mice after gavage
with BODIPY fatty acid analog. N=4-5. (H) Serum free fatty acid
levels in Mfge8+/+ and Mfge8-/- mice after a 24 hour fast, N=4-5.
(I-K) Effect of oral administration of integrin blocking antibodies
prior to olive oil gavage on serum triglycerides levels (G,
*P<0.01, **P<0.001, ***P<0.0001, when comparing .alpha.v
antibody with control antibody, .sup.#P<0.01 when comparing
.beta.5 antibody with control antibody), and on small intestine
(FIG. 3H) and liver triglyceride content (FIG. 3I) in Mfge8+/+
mice. N=4-5 for panels, I-K. .sup.#P<0.05, *P<0.01,
**P<0.001, ***P<0.0001 where not previously specified. Data
are expressed as mean.+-.s.e.m. Each in vivo experiment was
performed once. For in vitro experiments in panel B, each replicate
represents an independent experiment.
[0056] FIGS. 4A-4F. Mfge8 increases fatty acid uptake through an
integrin-PI3K-AKT-dependent pathway. (A) Effect of rMfge8 (10
.mu.g/mL) or insulin (10 .mu.g/mL) with and without wortmannin (100
nM) on AKT phosphorylation. (B) Effect of wortmannin on fatty acid
uptake in primary Mfge8+/+ and Mfge8-/- adipocytes and on Mfge8-/-
adipocytes treated with rMfge8. N=7. *P<0.05, ***P<0.001.
Data are expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment. (C) Effect of mutated Mfge8 constructs on
AKT and Rictor phosphorylation in 3T3-L1 adipocytes. (D) Effect of
integrin blocking antibodies on AKT phosphorylation in 3T3-L1
adipocytes after treatment with rMfge8. (E) The effect of mutated
Mfge8 constructs on phosphorylation of AKT and Rictor in HepG2
cells. (F) The effect of rMfge8 on AKT phosphorylation in the
presence of integrin blocking antibodies in HepG2 cells.
[0057] FIGS. 5A-5I. Mfge8 stimulates fatty acid uptake by inducing
translocation of fatty acid transporters to the cell surface. (A-C)
Plasma membrane and post membrane CD36 expression in primary
Mfge8-/- and Mfge8+/+ adipocytes (A), hepatocytes (B), and
enterocytes (C). Mfge8-/- cells were also incubated with rMfge8 or
rMfge8 and wortmannin. (D-F) Effect of CD36 blocking antibody or
control antibody on the ability of rMfge8 to increase fatty acid
uptake in primary Mfge8-/- and Mfge8+/+ adipocytes (D), hepatocytes
(E), and enterocytes (F), and on Mfge8+/+ adipocytes, hepatocytes,
and enterocytes. N=3-4 for experiments with antibodies and 7-8 for
experiments with and without rMfge8. (G) Effect of rMfge8 on fatty
acid uptake in CD36+/+ and CD36-/- adipocytes. N=3 for experiments
with CD36-/- cells and rMfge8 and 6 for experiments with Mfge8+/+
and Mfge8-/- cells, each replicate represents an independent
experiment and data from different experiments were combined for
statistical analysis. (H) Plasma membrane and post membrane
expression of FATP1 in primary Mfge8-/- and Mfge8+/+ adipocytes,
and Mfge8-/- adipocytes treated with rMfge8 or rMfge8 and
wortmannin. (I) Effect of rMfge8 on fatty acid uptake in FATP1+/+
and FATP1-/- adipocytes. N=3, each replicate represents an
independent experiment. *P<0.05, **P<0.001, ***P<0.0001.
Data are expressed as mean.+-.s.e.m.
[0058] FIG. 6. Effect of integrin blocking antibodies on fatty acid
uptake by Mfge8-/- adipocytes. Fatty acid uptake was measured in
Mfge8-/- adipocytes in the presence of integrin blocking antibodies
(20 .mu.g/ml). N=4. Data are expressed as mean.+-.s.e.m. Each
replicate represents an independent experiment.
[0059] FIGS. 7A-7C. Mfge8 does not regulate glucose homeostasis.
(A) Serum glucose levels after gavage with a glucose bolus in
Mfge8+/+ and Mfge8-/- mice and gavage of glucose mixed with rMfge8
in Mfge8-/- mice. N=4-5. (B) Effect of rMfge8 and insulin on
glucose uptake in 3T3-L1 adipocytes. N=7-8, each replicate
represents an independent experiment. (C) Effect of integrin
blocking antibodies or control antibody on glucose adsorption by
Mfge8+/+ mice after glucose gavage. N=4-5. Data are expressed as
mean.+-.s.e.m. Each replicate represents an independent experiment.
*P<0.01.
[0060] FIGS. 8A-8H demonstrates an effect of Mfge8 on gastric
motility. (A,B) Smooth muscle strips from stomach of Mfge KO
(knockout) mice have stronger contraction and thus can be rescued
by addition of rMfge8. (C) Represents the same experiment as FIGS.
13A and B but using mice that only express Mfge8 in smooth muscle
(dbl-means transgenic mice in the Mfge8 KO background that express
Mfge8 in smooth muscle), have no Mfge8 (single-sgl), or are wild
type. (D, E) Increased small intestinal transit time with rescue
groups as above in Mfge8 KO mice. (F, G) More rapid gastric
emptying in Mfge KO with same rescue as above. (H) Increased
phosphorylation of MLCP meaning enhanced calcium sensitivity in
Mfge8 KO mice.
[0061] FIG. 9 shows that Mfge8 induces insulin resistance in 3T3-L1
adipocytes. Glucose uptake in 3T3-L1 adipocyte with and without 20
min treatment with recombinant Mfge8 or RGE (10 .mu.g/ml) and
insulin (1 .mu.M) or both Mfge8 and insulin (n=8, P<0.05). Data
are expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment.
[0062] FIG. 10 illustrates that integrin receptor blockade enhances
insulin sensitivity in primary adipocytes. Glucose uptake in
Mfge8-/- and Mfge8+/+ primary adipocytes, with and without 20 min
treatment with insulin (1 .mu.M) and effect of pretreatment with
integrin blocking antibodies (0.5 .mu.g/g, IP, 15 min before
insulin) on glucose uptake in Mfge8+/+ adipocytes. Data are
expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment (n=8, P<0.05). Pretreatment with av, b3
or b5 integrin blocking antibody prior to insulin injection
resulted in significantly lower serum glucose levels after insulin
injection as compared with insulin injection alone.
[0063] FIG. 11 shows that Mfge8 induces acute insulin resistance in
vivo. 8-week-old Mfge8-/- and Mfge8+/+ control mice were fasted for
4 hours, then blood glucose was measured 15 min after IP injection
of insulin (1 U/kg), saline, RGE (50 .mu.g/kg) or a combination of
insulin (1.5 U/Kg) and rMfge8 or RGE construct (50 .mu.g/kg). Data
are expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment (n=4, P<0.05).
[0064] FIG. 12 shows that integrin blockade induces acute insulin
sensitivity in vivo. 8-week-old Mfge8-/- and Mfge8+/+ control mice
were fasted for 4 hours, then received blocking antibodies (0.5
.mu.g per gram body weight)(.alpha.v (clone RMV-7) and .beta.v
(clone ALULA)) IP. 15 min prior insulin (1 U/kg) or saline, blood
glucose was measured 15 min after IP injection of insulin (1 U/kg)
or saline. Data are expressed as mean.+-.s.e.m. Each replicate
represents an independent experiment (n=4, P<0.05).
[0065] FIGS. 13A-13G. Enhanced, antral smooth muscle contraction in
Mfge8-/- mice. (A-C) Force of antral smooth muscle ring contraction
with and without the addition of rMfge8 or RGE construct (A) or
after in vivo induction of transgenic Mfge8 expression in
Mfge8-/-sm+ mice in response to MCh. (C) Force of antral
contraction with and without epithelium (denuded). Mfge8-/- mice
have enhanced gastric emptying and more rapid small intestine
transit time (SIT). (D-E) Gastric emptying was measured by the
proportion of phenol red remaining in the stomach 15 minutes after
gavage. N=7-10. (F-G) Small intestinal transit times after gavage
with Carmine dye with subsequent evaluation at 15 minutes of dye
migration along the intestinal tract. N=5-10 in C and 3-5 in D. For
FIGS. 13E and 13G, Mfge8-/-sm+ and single transgenic controls were
placed on doxycycline for 2 weeks prior to the experiments to
induce Mfge8 production in the smooth muscle. *P<0.05,
**P<0.01, ***P<0.001.
[0066] FIGS. 14A-14D. Inhibition of PI3K prevents exaggerated
Mfge8-/- antral ring contraction. (A) Antral rings were treated for
15 min with PI3K inhibitor wortmannin (Wort. 100 nm) followed by
assessment of contractile force in response to MCh. N=3-4.
***P<0.0001. (B) Mfge8 reduces AKT phosphorylation. Western blot
of antral tissue treated for 30 minutes with or without rMfge8. (C)
Wortmannin prevents RhoA activation. Western blot of antrum treated
for 30 minutes with wortmannin (100 ng/ml) or Mfge8 and then with
MCh for 15 minutes prior to quantifying active RhoA using a GST
pull-down. (D) Mfge8 modulates PTEN activity. PTEN activity assay
measuring conversion of PIP3 to PIP2 in freshly isolated antrum
with and without rMfge8 or RGE construct (10 .mu.g/ml) n=3-5.
*P<0.05, **P<0.01, ***P<0.001.
DETAILED DESCRIPTION OF THE INVENTION
[0067] I. Introduction
[0068] Surprisingly, it has been discovered that Milk Fat Globule
Epidermal Growth Factor 8 (Mfge8) stimulates fatty acid uptake. In
view of this discovery, Mfge8 and analogs thereof will be useful in
stimulating fatty uptake in animals (including but not limited to
humans) in need thereof. Individuals who will benefit from
stimulated fatty acid uptake include but are not limited to
premature infants, individuals with excessive gastric emptying,
diabetics and those with cystic fibrosis.
[0069] Conversely, blocking Mfge8 binding to integrin receptors
.alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5 inhibits
fatty acid uptake. Blocking fatty acid uptake is useful, for
example, in weight loss. Accordingly, administration of antagonists
of .alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5 is useful
for inducing weight loss in animals (e.g., humans), and is
particularly useful for obese individuals. In addition,
administration of antagonists of .alpha..sub.v.beta..sub.3 or
.alpha..sub.v.beta..sub.5 can also enhance insulin sensitivity and
can be used to treat individuals with reduced insulin sensitivity
or insulin resistance.
[0070] II. Increasing MFGE8 Signaling
[0071] As noted above, it has been discovered that Mfge8 increases
fatty acid uptake in various organs. Accordingly, in some
embodiments, a polypeptide comprising Mfge8, or a substantially
identical sequence thereof, or an integrin-binding fragment thereof
is administered to an animal, thereby increasing fatty acid uptake
in the animal. In some embodiments, a Mfge8 polypeptide as
described herein is administered to an individual suffering from
fat malabsorption.
[0072] In some aspects, an individual in need of increased fatty
acid uptake/adsorption is treated with a Mfge8 polypeptide as
described herein. In some aspects, the individual has excess
gastric emptying or otherwise has difficulty extracting nutrients
from food. In some embodiments, the individual is a premature
infant (e.g., born prior to completion of gestation) or is
otherwise nursing. In some embodiments, the individual is less than
1 or 2 years old. In some embodiments, the individual is less than
6, 5, 4, 3, 2, or 1 month old. Additional examples of individuals
(e.g., suffering from fat malabsorption) to which Mfge8
polypeptides can be administered include individuals having cystic
fibrosis, individuals who undergo gastric bypass surgery,
individuals with a decrease in the small intestine lymphatics,
individuals having altered duodenal pH (Zollinger-Ellison
syndrome), individuals having improper emulsification after certain
types of gastrectomy, individuals having rapid transit dumping
syndrome, rapid transit dumping syndrome having acute abnormality
in the intestinal lining, e.g., because of infections, antibiotics
or alcohol abuse, rapid transit dumping syndrome having small bowel
syndrome, rapid transit dumping syndrome having presence of a
chronic abnormal intestinal lining, e.g., as a result of conditions
such as Crohn's disease or Celiac disease, rapid transit dumping
syndrome having improper intestinal environments, e.g., because of
bacterial overgrowth or the presence of parasites in the digestive
system, rapid transit dumping syndrome having inadequate gastric
mixing, e.g., due to factors such as a fistula in the gastric
environment or after a gastrostomy, rapid transit dumping syndrome
having impaired movement of the enzymes in the body, individuals
having intestinal lymphangiectasia, individuals having Whipple's
Disease, rapid transit dumping syndrome having irritable bowel
syndrome and/or inflammatory bowel disease, individuals having
cancer (e.g., advanced cancer such as Stage III or Stage IV). A
Mfge8 polypeptide as described herein is administered to an
individual having pancreatic and biliary dysfunction, including but
not limited to individuals having chronic pancreatitis, obstruction
in the pancreatic duct, pancreatic cancer, resection of the
pancreas, Shwachmann-Diamond syndrome, Johnson-Blizzard syndrome,
or Pearson syndrome.
[0073] A variety of polypeptides can be used according to the
methods for increasing fatty acid uptake. In some embodiments,
full-length native (or variants thereof) Mfge8 protein amino acid
sequences are used in the methods described herein. In some
embodiments, the polypeptides comprise a fragment (not the
full-length native sequence) of the Mfge8 sequence, or a variant
thereof, that retains the ability to bind to integrin
.alpha..sub.v.beta..sub.3 or .alpha..sub.v.beta..sub.5. Binding of
Mfge8 to the integrin receptors is through the RGD motif and thus
in some embodiments the Mfge8 fragment will comprise at least RGD,
and in some aspects at least 2, 4, 5, 10, or 20 native amino acids
of Mfge8 on either side of the RGD motif. Thus, in some
embodiments, the polypeptides comprise at least a fragment (e.g.,
at least 20, 40, 50, 100, 150, 200, 250 contiguous amino acids) of
the native Mfge8 protein. The inventors have found that the
sequence set forth in SEQ ID NO:4, which is a mouse Mfge8 sequence
lacking the second discoidan domain but including all other native
sequence, remains active. The full length mouse Mfge8 sequence is
depicted in SEQ ID NO:3.
[0074] In some aspects the Mfge8 protein or fragment thereof will
be derived from the animal species to be treated. Thus, for
example, if a human is to be treated, a human Mfge8 (e.g., SEQ ID
NO:1) or fragment thereof is administered. As another example, if
cattle are to be treated, a bovine Mfge8 (e.g., SEQ ID NO:5) or a
fragment or substantially identical polypeptide thereof is
used.
[0075] While not required, in some embodiments, the Mfge8
polypeptides will include non-native Mfge8 protein flanking
sequences. For example, a full-length Mge8 or an RGD-containing
fragment of Mfge8 can be fused to one or more heterologous amino
acids to form a fusion protein. Fusion partner sequences can
include, but are not limited to, amino acid tags, non-L (e.g., D-)
amino acids or other amino acid mimetics to extend in vivo
half-life and/or protease resistance, targeting sequences or other
sequences.
[0076] In some embodiments, the MFGE8 polypeptides will comprise at
least one non-naturally encoded amino acid. Methods of making and
introducing a non-naturally-occurring amino acid into a protein are
known. See, e.g., U.S. Pat. Nos. 7,083,970; and 7,524,647. The
general principles for the production of orthogonal translation
systems that are suitable for making proteins that comprise one or
more desired unnatural amino acid are known in the art, as are the
general methods for producing orthogonal translation systems. For
example, see International Publication Numbers WO 2002/086075,
entitled "METHODS AND COMPOSITION FOR THE PRODUCTION OF ORTHOGONAL
tRNA-AMINOACYL-tRNA SYNTHETASE PAIRS;" WO 2002/085923, entitled "IN
VIVO INCORPORATION OF UNNATURAL AMINO ACIDS;" WO 2004/094593,
entitled "EXPANDING THE EUKARYOTIC GENETIC CODE;" WO 2005/019415,
filed Jul. 7, 2004; WO 2005/007870, filed Jul. 7, 2004; WO
2005/007624, filed Jul. 7, 2004; WO 2006/110182, filed Oct. 27,
2005, entitled "ORTHOGONAL TRANSLATION COMPONENTS FOR THE VIVO
INCORPORATION OF UNNATURAL AMINO ACIDS" and WO 2007/103490, filed
Mar. 7, 2007, entitled "SYSTEMS FOR THE EXPRESSION OF ORTHOGONAL
TRANSLATION COMPONENTS IN EUBACTERIAL HOST CELLS." Each of these
applications is incorporated herein by reference in its entirety.
For discussion of orthogonal translation systems that incorporate
unnatural amino acids, and methods for their production and use,
see also, Wang and Schultz, (2005) "Expanding the Genetic Code."
Angewandte Chemie Int Ed 44: 34-66; Xie and Schultz, (2005) "An
Expanding Genetic Code." Methods 36: 227-238; Xie and Schultz,
(2005) "Adding Amino Acids to the Genetic Repertoire." Curr Opinion
in Chemical Biology 9: 548-554; and Wang, et al., (2006) "Expanding
the Genetic Code." Annu Rev Biophys Biomol Struct 35: 225-249;
Deiters, et al, (2005) "In vivo incorporation of an alkyne into
proteins in Escherichia coli." Bioorganic & Medicinal Chemistry
Letters 15:1521-1524; Chin, et al., (2002) "Addition of
p-Azido-L-phenylalanine to the Genetic Code of Escherichia coli." J
Am Chem Soc 124: 9026-9027; and International Publication No.
WO2006/034332, filed on Sep. 20, 2005, the contents of each of
which are incorporated by reference in their entirety. Additional
details are found in U.S. Pat. No. 7,045,337; U.S. Pat. No.
7,083,970; U.S. Pat. No. 7,238,510; U.S. Pat. No. 7,129,333; U.S.
Pat. No. 7,262,040; U.S. Pat. No. 7,183,082; U.S. Pat. No.
7,199,222; and U.S. Pat. No. 7,217,809.
[0077] A "non-naturally encoded amino acid" refers to an amino acid
that is not one of the common amino acids or pyrolysine or
selenocysteine. Other terms that may be used synonymously with the
term "non-naturally encoded amino acid" are "non-natural amino
acid," "unnatural amino acid," "non-naturally-occurring amino
acid," and variously hyphenated and non-hyphenated versions
thereof. The term "non-naturally encoded amino acid" also includes,
but is not limited to, amino acids that occur by modification (e.g.
post-translational modifications) of a naturally encoded amino acid
(including but not limited to, the 20 common amino acids or
pyrrolysine and selenocysteine) but are not themselves naturally
incorporated into a growing polypeptide chain by the translation
complex. Examples of such non-naturally-occurring amino acids
include, but are not limited to, N-acetylglucosaminyl-L-serine,
N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.
[0078] A non-naturally encoded amino acid is typically any
structure having any substituent side chain other than one used in
the twenty natural amino acids. Because the non-naturally encoded
amino acids of the invention typically differ from the natural
amino acids only in the structure of the side chain, the
non-naturally encoded amino acids form amide bonds with other amino
acids, including but not limited to, natural or non-naturally
encoded, in the same manner in which they are formed in naturally
occurring polypeptides. However, the non-naturally encoded amino
acids have side chain groups that distinguish them from the natural
amino acids. For example, R optionally comprises an alkyl-, aryl-,
acyl-, keto-, azido-, hydroxyl-, hydrazine, cyano-, halo-,
hydrazide, alkenyl, alkynl, ether, thiol, seleno-, sulfonyl-,
borate, boronate, phospho, phosphono, phosphine, heterocyclic,
enone, imine, aldehyde, ester, thioacid, hydroxylamine, amino
group, or the like or any combination thereof. Other non-naturally
occurring amino acids of interest that may be suitable for use in
the present invention include, but are not limited to, amino acids
comprising a photoactivatable cross-linker, spin-labeled amino
acids, fluorescent amino acids, metal binding amino acids,
metal-containing amino acids, radioactive amino acids, amino acids
with novel functional groups, amino acids that covalently or
noncovalently interact with other molecules, photocaged and/or
photoisomerizable amino acids, amino acids comprising biotin or a
biotin analogue, glycosylated amino acids such as a sugar
substituted serine, other carbohydrate modified amino acids,
keto-containing amino acids, amino acids comprising polyethylene
glycol or polyether, heavy atom substituted amino acids, chemically
cleavable and/or photocleavable amino acids, amino acids with an
elongated side chains as compared to natural amino acids, including
but not limited to, polyethers or long chain hydrocarbons,
including but not limited to, greater than about 5 or greater than
about 10 carbons, carbon-linked sugar-containing amino acids,
redox-active amino acids, amino thioacid containing amino acids,
and amino acids comprising one or more toxic moiety.
[0079] Exemplary non-naturally encoded amino acids that may be
suitable for use in the present invention and that are useful for
reactions with water soluble polymers include, but are not limited
to, those with carbonyl, aminooxy, hydrazine, hydrazide,
semicarbazide, azide and alkyne reactive groups. In some
embodiments, non-naturally encoded amino acids comprise a
saccharide moiety. Examples of such amino acids include
N-acetyl-L-glucosaminyl-L-serine,
N-acetyl-L-galactosaminyl-L-serine,
N-acetyl-L-glucosaminyl-L-threonine,
N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.
Examples of such amino acids also include examples where the
naturally-occurring N- or O-linkage between the amino acid and the
saccharide is replaced by a covalent linkage not commonly found in
nature--including but not limited to, an alkene, an oxime, a
thioether, an amide and the like. Examples of such amino acids also
include saccharides that are not commonly found in
naturally-occurring proteins such as 2-deoxy-glucose,
2-deoxygalactose and the like.
[0080] Another type of modification that can optionally be
introduced into the MFGE8 polypeptide (e.g., within the polypeptide
chain or at either the N- or C-terminal), e.g., to extend in vivo
half-life, is PEGylation or incorporation of long-chain
polyethylene glycol polymers (PEG). Introduction of PEG or long
chain polymers of PEG increases the effective molecular weight of
the present polypeptides, for example, to prevent rapid filtration
into the urine. In some embodiments, a Lysine residue in the MFGE8
sequence is conjugated to PEG directly or through a linker. Such
linker can be, for example, a Glu residue or an acyl residue
containing a thiol functional group for linkage to the
appropriately modified PEG chain. An alternative method for
introducing a PEG chain is to first introduce a Cys residue at the
C-terminus or at solvent exposed residues such as replacements for
Arg or Lys residues. This Cys residue is then site-specifically
attached to a PEG chain containing, for example, a maleimide
function. Methods for incorporating PEG or long chain polymers of
PEG are well known in the art (described, for example, in Veronese,
F. M., et al., Drug Disc. Today 10: 1451-8 (2005); Greenwald, R.
B., et al., Adv. Drug Deliv. Rev. 55: 217-50 (2003); Roberts, M.
J., et al., Adv. Drug Deliv. Rev., 54: 459-76 (2002)), the contents
of which is incorporated herein by reference. Other methods of
polymer conjugations known in the art can also be used in the
present invention. In some embodiments, poly(2-methacryloyloxyethyl
phosphorylcholine) (PMPC) is introduced as a polymer conjugate with
the MFGE8 proteins of the invention (see, e.g., WO2008/098930;
Lewis, et al., Bioconjug Chem., 19: 2144-55 (2008)). In some
embodiments, a phosphorylcholine-containing polymer conjugate with
the MFGE8 proteins can be used in the present invention. Other
biocompatible polymer conjugates can also be utilized.
[0081] A more recently reported alternative approach for
incorporating PEG or PEG polymers through incorporation of
non-natural amino acids (as described above) can be performed with
the present Mfge8 polypeptides. This approach utilizes an evolved
tRNA/tRNA synthetase pair and is coded in the expression plasmid by
the amber suppressor codon (Deiters, A, et al. (2004). Bio-org.
Med. Chem. Lett. 14, 5743-5). For example, p-azidophenylalanine can
be incorporated into the present polypeptides and then reacted with
a PEG polymer having an acetylene moiety in the presence of a
reducing agent and copper ions to facilitate an organic reaction
known as "Huisgen [3+2]cycloaddition."
[0082] In certain embodiments, specific mutations of the MFGE8
proteins are contemplated so as to alter the glycosylation of the
polypeptide. Such mutations may be selected so as to introduce or
eliminate one or more glycosylation sites, including but not
limited to, O-linked or N-linked glycosylation sites. In certain
embodiments, the MFGE8 proteins have glycosylation sites and
patterns unaltered relative to the naturally-occurring MFGE8
proteins. In certain embodiments, a variant of MFGE8 proteins
includes a glycosylation variant wherein the number and/or type of
glycosylation sites have been altered relative to the
naturally-occurring MFGE8 proteins. In certain embodiments, a
variant of a polypeptide comprises a greater or a lesser number of
N-linked glycosylation sites relative to a native polypeptide. An
N-linked glycosylation site is characterized by the sequence:
Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated
as X may be any amino acid residue except proline. The substitution
of amino acid residues to create this sequence provides a potential
new site for the addition of an N-linked carbohydrate chain.
Alternatively, substitutions which eliminate this sequence will
remove an existing N-linked carbohydrate chain. In certain
embodiments, a rearrangement of N-linked carbohydrate chains is
provided, wherein one or more N-linked glycosylation sites
(typically those that are naturally occurring) are eliminated and
one or more new N-linked sites are created.
[0083] Exemplary MFGE8 proteins variants include cysteine variants
wherein one or more cysteine residues are deleted from or
substituted for another amino acid (e.g., serine) relative to the
amino acid sequence of the naturally-occurring MFGE8 proteins. In
certain embodiments, cysteine variants may be useful when MFGE8
proteins must be refolded into a biologically active conformation
such as after the isolation of insoluble inclusion bodies. In
certain embodiments, cysteine variants have fewer cysteine residues
than the native polypeptide. In certain embodiments, cysteine
variants have an even number of cysteine residues to minimize
interactions resulting from unpaired cysteines.
[0084] In some embodiments, functional variants or modified forms
of the MFGE8 proteins include fusion proteins of an MFGE8 protein
of the invention and one or more fusion domains. Well known
examples of fusion domains include, but are not limited to,
polyhistidine, Glu-Glu, glutathione S transferase (GST),
thioredoxin, protein A, protein G, an immunoglobulin heavy chain
constant region (Fc), maltose binding protein (MBP), or human serum
albumin. A fusion domain may be selected so as to confer a desired
property. For example, some fusion domains are particularly useful
for isolation of the fusion proteins by affinity chromatography.
For the purpose of affinity purification, relevant matrices for
affinity chromatography, such as glutathione-, amylase-, and
nickel- or cobalt-conjugated resins are used. Many of such matrices
are available in "kit" form, such as the Pharmacia GST purification
system and the QLAexpress.TM. system (Qiagen) useful with fusion
partners (e.g., His.sub.6; SEQ ID NO:7). As another example, a
fusion domain may be selected so as to facilitate detection of the
MFGE8 proteins. Examples of such detection domains include the
various fluorescent proteins (e.g., GFP) as well as "epitope tags,"
which are usually short peptide sequences for which a specific
antibody is available. Well known epitope tags for which specific
monoclonal antibodies are readily available include FLAG, influenza
virus haemagglutinin (HA), and c-myc tags. In some cases, the
fusion domains have a protease cleavage site, such as for Factor Xa
or Thrombin, which allows the relevant protease to partially digest
the fusion proteins and thereby liberate the recombinant proteins
therefrom. The liberated proteins can then be isolated from the
fusion domain by subsequent chromatographic separation. In certain
embodiments, an MFGE8 protein is fused with a domain that
stabilizes the MFGE8 protein in vivo (a "stabilizer" domain). By
"stabilizing" is meant anything that increases serum half life,
regardless of whether this is because of decreased destruction,
decreased clearance by the kidney, or other pharmacokinetic effect.
Fusions with the Fc portion of an immunoglobulin are known to
confer desirable pharmacokinetic properties on a wide range of
proteins. Likewise, fusions to human serum albumin can confer
desirable properties. Other types of fusion domains that may be
selected include multimerizing (e.g., dimerizing, tetramerizing)
domains and functional domains (that confer an additional
biological function, as desired).
[0085] It is contemplated that the polypeptides, compositions, and
methods of the present invention may be used to treat a mammal. As
used herein a "mammal" to any mammal classified as a mammal,
including humans, domestic and farm animals, and zoo, sports or pet
animals, such as cattle (e.g. cows), horses, dogs, sheep, pigs,
rabbits, goats, cats, etc. All embodiments described herein not
specifically referring to another species should be understood to
specifically apply to humans as well as more generally to
mammals.
[0086] The dose of a compound of the present invention for treating
the above-mentioned diseases or disorders varies depending upon the
manner of administration, the age and the body weight of the
subject, and the condition of the subject to be treated, and
ultimately will be decided by the attending physician or
veterinarian. Such an amount of the compound as determined by the
attending physician or veterinarian is referred to herein as an
"effective amount."
[0087] Formulations suitable for administration include excipients,
including but not limited to, aqueous and non-aqueous solutions,
isotonic sterile solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. In some embodiments, the Mfge8
polypeptides are formulated in micelles or liposomes.
[0088] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
response in the subject over time. The dose will be determined by
the efficacy of the particular protein employed and the condition
of the subject, as well as the body weight or surface area of the
area to be treated. The size of the dose also will be determined by
the existence, nature, and extent of any adverse side-effects that
accompany the administration of a particular protein or vector in a
particular subject. Administration can be accomplished via single
or divided doses.
[0089] Formulations suitable for administration include aqueous and
non-aqueous solutions, isotonic sterile solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, orally, rectally, nasally, topically, intravenously,
intraperitoneally, or intrathecally. The formulations of
polypeptides can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials. Solutions and suspensions
can be prepared from sterile powders, granules, and tablets of the
kind previously described. The modulators can also be administered
as part a of prepared food or drug.
[0090] In general, any food or beverage that can be consumed by
human infants or adults or animals may be used to make formulations
containing the Mfge8 polypeptides described herein. Exemplary foods
include those with a semi-liquid consistency to allow easy and
uniform dispersal of the compositions. However, other consistencies
(e.g., powders, liquids, etc.) can also be used without limitation.
Accordingly, such food items include, without limitation,
dairy-based products such as cheese, cottage cheese, yogurt, and
ice cream. Processed fruits and vegetables, including those
targeted for infants/toddlers, such as apple sauce or strained
vegetables (e.g., peas and carrots, etc.), are also suitable for
use in combination with the prebiotic and synbiotic compositions of
the present invention. Both infant cereals such as rice- or
oat-based cereals and adult cereals such as Musilix are also
suitable for use in combination with the oligosaccharides of the
present invention. In addition to foods targeted for human
consumption, animal feeds may also be supplemented with the Mfge8
polypeptides as described herein.
[0091] Alternatively, the Mfge8 polypeptide compositions can be
used to supplement a beverage. Examples of such beverages include,
without limitation, infant formula, follow-on formula, toddler's
beverage, milk, fermented milk, fruit juice, fruit-based drinks,
and sports drinks. Many infant and toddler formulas are known in
the art and are commercially available, including, for example,
Carnation Good Start (Nestle Nutrition Division; Glendale, Calif.)
and Nutrish A/B produced by Mayfield Dairy Farms (Athens, Tenn.).
Other examples of infant or baby formula include those disclosed in
U.S. Pat. No. 5,902,617. Other beneficial formulations of the
compositions of the present invention include the supplementation
of animal milks, such as cow's milk.
[0092] Alternatively, the prebiotic and probiotic compositions of
the present invention can be formulated into pills or tablets or
encapsulated in capsules, such as gelatin capsules. Tablet forms
can optionally include, for example, one or more of lactose,
sucrose, mannitol, sorbitol, calcium phosphates, corn starch,
potato starch, microcrystalline cellulose, gelatin, colloidal
silicon dioxide, talc, magnesium stearate, stearic acid, and other
excipients, colorants, fillers, binders, diluents, buffering
agents, moistening agents, preservatives, flavoring agents, dyes,
disintegrating agents, and pharmaceutically compatible carriers.
Lozenge or candy forms can comprise the compositions in a flavor,
e.g., sucrose, as well as pastilles comprising the compositions in
an inert base, such as gelatin and glycerin or sucrose and acacia
emulsions, gels, and the like containing, in addition to the active
ingredient, carriers known in the art. The Mfge8 polypeptide
formulations may also contain conventional food supplement fillers
and extenders such as, for example, rice flour.
[0093] In some embodiments, the Mfge8 polypeptide composition will
further comprise a non-human protein, non-human lipid, non-human
carbohydrate, or other non-human component. For example, in some
embodiments, the compositions of the invention comprise a bovine
(or other non-human) milk protein, a soy protein, a rice protein,
betalactoglobulin, whey, soybean oil or starch. In some
embodiments, the prebiotic or synbiotic composition will further
comprise a non-bovine protein, non-bovine lipid, non-bovine
carbohydrate, or other non-bovine component.
[0094] III. Inhibiting MFGE8 Signaling
[0095] Mfge8 is a ligand for both .alpha..sub.v.beta..sub.3 and
.alpha..sub.v.beta..sub.5 integrins. As demonstrated below in
Examples 1 and 2, administration of an antagonist antibody that
binds the .alpha.v integrin subunit completely inhibited fat uptake
in an animal and administration of an antibody specific for
.alpha..sub.v.beta..sub.3 or administration of an antibody specific
for .alpha..sub.v.beta..sub.5 partially decreases fat absorption.
Accordingly, antagonizing the .alpha..sub.v.beta..sub.3 and/or
.alpha..sub.v.beta..sub.5 integrin receptors is effective to reduce
fat uptake and optionally for weight reduction in an
individual.
[0096] Co-administration of antagonists of
.alpha..sub.v.beta..sub.3 and/or .alpha..sub.v.beta..sub.5 integrin
receptors and insulin can be used to treat individuals with reduced
insulin sensitivity. In particular, pretreatment with antagonists
of .alpha..sub.v.beta..sub.3 and/or .alpha..sub.v.beta..sub.5
integrin receptors prior to insulin injection can significantly
lower serum glucose levels, as compared to injection of insulin
alone.
[0097] Exemplary antagonists can be, for example, antagonist
antibodies, e.g., antagonists antibodies that specifically bind to
.alpha..sub.v.beta..sub.3 and/or .alpha..sub.v.beta..sub.5 integrin
receptors. An exemplary .alpha..sub.v.beta..sub.5 antibody is
"ALULA" (the antibody produced by the hybridoma deposited under
ATCC Deposit No. PTA-5817, made Feb. 13, 2004, at the ATCC, 10801
University Blvd. Manassas, Va. 20110-2209). Alternatively, the
.alpha..sub.v.beta..sub.3 and/or .alpha..sub.v.beta..sub.5 integrin
receptor antagonists can be small molecules. See, e.g., PCT WO
2003/059872. In some embodiments, the antagonists are
RGD-containing peptides or cyclic peptides. Examples of such
peptides include, but are not limited to those described in
Belvisi, L., et al., Mol Cancer Ther 4(11): (Nov. 2005). Some
antagonists are reviewed in Hsu, A R, et al., Recent Pat Anticancer
Drug Discov. 2(2):143-58 (2007).
[0098] In some embodiments, the .alpha..sub.v.beta..sub.3 and/or
.alpha..sub.v.beta..sub.5 integrin receptor antagonists are
administered to an individual that is overweight overweight (e.g.,
body mass index (BMI) greater or equal to 25 kg/m.sup.2) or is
obese. In some embodiments, the .alpha..sub.v.beta..sub.3 and/or
.alpha..sub.v.beta..sub.5 integrin receptor antagonists are
administered to an individual with high (i.e., above average) level
of fatty acids and triglycerides, and an individual with
hyperlipidemia or dyslipidemia (e.g., an individual having
cirrhosis or liver damage, hypothyroidism (underactive thyroid),
nephrotic syndrome, a kidney disorder, or diabetes. In some
embodiments, the .alpha..sub.v.beta..sub.3 and/or
.alpha..sub.v.beta..sub.5 integrin receptor antagonists are
administered to a pregnant individual or an individual with
elevated levels of female hormones. In some embodiments, the
.alpha..sub.v.beta..sub.3 and/or .alpha..sub.v.beta..sub.5 integrin
receptor antagonists are administered to an individual with an
elevated risk cardiovascular disease or stroke. In some
embodiments, the .alpha..sub.v.beta..sub.3 and/or
.alpha..sub.v.beta..sub.5 integrin receptor antagonists are
administered to an individual with reduced insulin sensitivity or
insulin resistance.
[0099] The .alpha..sub.v.beta..sub.3 and/or
.alpha..sub.v.beta..sub.5 integrin receptor antagonists can be
formulated and administered as described above with regard to the
Mfge8 polypeptides. For example, formulations suitable for
administration include aqueous and non-aqueous solutions, isotonic
sterile solutions, which can contain antioxidants, buffers,
bacteriostats, and solutes that render the formulation isotonic,
and aqueous and non-aqueous sterile suspensions that can include
suspending agents, solubilizers, thickening agents, stabilizers,
and preservatives. In the practice of this invention, compositions
can be administered, for example, orally, rectally, nasally,
topically, intravenously, intraperitoneally, or intrathecally. The
formulations of polypeptides can be presented in unit-dose or
multi-dose sealed containers, such as ampoules and vials. Solutions
and suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described. The modulators can also
be administered as part of a prepared food or drug.
EXAMPLES
Example 1: Mfge8 and Regulating Fat Storage and Lipid
Homeostasis
[0100] Fatty acids are integral mediators of energy storage,
membrane formation, and cell signaling. The pathways that
orchestrate uptake of fatty acids remain incompletely understood.
Expression of the integrin ligand Mfge8 is increased in human
obesity and in mice on a high-fat diet (HFD). The role of Mfge8 in
obesity is unknown. We show here that Mfge8 promotes the
development of obesity by facilitating cellular uptake of fatty
acids.
[0101] Mfge8 deficient (Mfge8-/-) mice absorb less dietary
triglyerides and are protected from weight gain, steatohepatitis
and obesity-associated insulin resistance on a HFD. Mfge8-/- cells
have impaired fatty acid uptake in vitro. Mfge8 coordinates fatty
acid uptake through .alpha.v.beta.3 and .alpha.v.beta.5
integrin-dependent phosphorylation of AKT by PI3 kinase and mTOR
complex 2 leading to translocation of CD36 and FATP1 from cytosolic
stores to the cell surface. Collectively, our results implicate a
central role for Mfge8 in regulating fat storage and lipid
homeostasis.
[0102] Results
Mfge8 Increases 3T3-L1 Adipocyte Fatty Acid Uptake and Triglyceride
Storage
[0103] To evaluate the effect of Mfge8 on fatty acid uptake, we
quantified the effect of recombinant Mfge8 (rMfge8) on uptake of a
BODIPY fatty acid analog (Liao, J. et al., J Lipid Res 46, 597-602
(2005)) by 3T3-L1 adipocytes. rMfge8 significantly increased fatty
acid uptake in a dose-dependent fashion (FIG. 1A), while a
recombinant construct with a point mutation changing the
integrin-binding RGD sequence of Mfge8 to RGE (RGE) had no effect
(FIG. 1A). 3T3-L1 cells treated with rMfge8, but not RGE, had
significantly greater triglyceride content 2, 4, 6, and 8 days
after treatment (FIG. 1B). These data indicate that Mfge8 increases
fatty acid uptake and triglyceride stores in 3T3-L1 adipocytes
through an integrin-dependent pathway.
Mfge8-/- Adipocytes have Impaired Fatty Acid Uptake that is Rescued
with rMfge8.
[0104] We next evaluated whether fatty acid uptake was impaired in
adipocytes from Mfge8-/- mice. Mfge8-/- primary adipocytes isolated
from epididymal white adipose tissue (eWAT) and differentiated
adipocyte progenitors isolated from subcutaneous white adipose
tissue had significantly impaired fatty acid uptake (FIGS. 1C,D).
rMfge8 rescued the decrease in fatty acid uptake in Mfge8-/-
adipocytes and significantly increased WT fatty acid uptake (FIGS.
1C,D). In addition to an intact integrin-binding motif, the effect
of recombinant protein required at least one of the discoidin
domains of Mfge8 (FIGS. 1C,E,F). Treatment of 323-L1 adipocytes
with cycloRGD did not induce an increase in AKT phosphorylation or
fatty acid uptake.
The Effect of Mfge8 on Fatty Acid Uptake is Mediated Through the
.alpha.v.beta.5 and .alpha.v.beta.3 Integrins.
[0105] Mfge8 is a ligand for the .alpha.v.beta.3 and
.alpha.v.beta.5 integrins (Hanayama, R. et al., Nature 417, 182-187
(2002)). To determine whether Mfge8 mediated fatty acid uptake
through these integrins, we evaluated the effects of
integrin-blocking antibodies on the ability of rMfge8 to rescue
impaired fatty acid uptake in Mfge8-/- adipocytes (FIG. 1G) and
increase fatty acid uptake in 3T3-L1 adipocytes (FIG. 1H). Blocking
antibody to the .alpha.v integrin subunit or both the .beta.3 and
.beta.5 subunits completely inhibited and blocking antibodies to
the .beta.5 or the .beta.3 integrin subunits partially inhibited
the increase in fatty acid uptake induced by rMfge8 (FIGS. 1G,H).
During 3T3-L1 differentiation from fibroblasts into adipocytes,
Mfge8 expression increased, while expression of av, .beta.3 and
.beta.5 integrin subunits was stably persistent. .beta.5-/- and
.beta.3-/- adipocytes had impaired fatty acid uptake which was
further reduced with the addition of blocking antibody to the
.beta.3 integrin subunit in .beta.5-/- adipocytes and vice versa.
These data indicate that the .alpha.v.beta.3 and .alpha.v.beta.5
integrins mediate the effect of Mfge8 on fatty acid uptake.
Mfge8 Regulates Hepatic and Cardiac Fatty Acid Uptake
[0106] To determine whether the effect of Mfge8 on fatty acid
uptake could be generalized to other tissues, we evaluated fatty
acid uptake in hepatocytes and cardiac myocytes. Mfge8-/-
hepatocytes and cardiac myocytes had impaired fatty acid uptake
that was rescued with rMfge8 (FIGS. 2A,B). rMfge8 also increased
fatty acid uptake in HepG2 cells, a human hepatocellular carcinoma
cell line, in an integrin binding and discoidin domain dependent
manner (FIGS. 2C,D).
Mfge8 Mediates Fatty Acid Uptake and Absorption of Dietary
Triglycerides in the Gastrointestinal Tract.
[0107] The high expression of Mfge8 in breast milk (Newburg, D. S.
et al., Lancet 351, 1160-1164 (1998)) led us to investigate a role
for Mfge8 in intestinal fat absorption. We found significantly
reduced small intestinal triglyceride content in Mfge8-/- mice
(FIG. 3A) and reduced in vitro fatty acid uptake by Mfge8-/-
primary enterocytes (FIG. 3B). Mfge8-/- mice had significantly
lower serum triglyceride levels after olive oil gavage. Adding
rMfge8, but not RGE, to olive oil significantly raised serum
triglyceride levels (FIG. 3C). Liver triglyceride levels after
gavage were significantly lower in Mfge8-/- mice and increased
significantly with rMfge8 treatment (FIG. 3D). Serum fatty acid
levels after olive oil gavage were also lower in Mfge8-/- mice
(FIG. 3E). rMfge8 significantly increased WT serum triglyceride
levels after olive oil gavage (FIG. 3F). There was no difference in
serum glucose levels after glucose gavage when comparing Mfge8-/-
and WT mice or in glucose uptake by 3T3-L1 adipocytes treated with
rMfge8. To prove that Mfge8 deficiency led to fatty acid
malabsorption, we administered a gavage of BODIPY fatty acid analog
and measured fecal BODIPY content. Mfge8-/- mice had significantly
greater fecal BODIPY levels (FIG. 3G). To separate the effects of
impaired enteral absorption of fatty acids from impaired peripheral
uptake of fatty acids on serum fatty acid levels, we measured serum
free fatty acids after fasting mice for 24 hours. Mfge8-/- mice had
significantly higher serum free fatty acids (FIG. 3H) indicating a
defect in peripheral uptake of fatty acids after starvation-induced
lipolysis.
[0108] We next evaluated the ability of enteral integrin blockade
to prevent fat absorption in WT mice. The administration of
.alpha.v-blocking or .beta.5-blocking antibody by gavage 30 minutes
prior to receiving an olive oil bolus significantly reduced serum
triglyceride levels, enterocyte triglyceride content, and hepatic
triglyceride content (FIGS. 3I-K) while having no effect on glucose
absorption (FIG. 7C).
Mfge8 Stimulates Fatty Acid Uptake Through a PI3
Kinase-mTORC2-AKT-Dependent Pathway.
[0109] PI3 kinase and AKT are integral parts of regulatory pathways
in metabolism (Manning, B. D. & Cantley, L. C., Cell 129,
1261-1274 (2007); Li, X. et al., Nature 447, 1012-1016 (2007)). We
therefore investigated whether the Mfge8-induced increase in fatty
acid uptake was mediated through an AKT/PI3K axis. rMfge8 induced
phosphorylation of AKT at serine 473 (s473) in 3T3-L1 cells and
this effect was completely blocked by the PI3 kinase inhibitor
wortmannin (FIG. 4A). Wortmannin also inhibited the ability of
rMfge8 to increase fatty acid uptake in Mfge8-/- adipocytes treated
with rMfge8 (FIG. 4B). AKT phosphorylation was dependent on an
intact integrin binding motif and the presence of at least one
discoidin domain (FIG. 4C) and was inhibited by blocking antibodies
to the .beta.v, .beta.3, and .beta.5 integrin subunits (FIG. 4D).
mTOR Complex 2 (mTORC2) is the kinase complex primarily responsible
for phosphorylation of AKT at s473 (Sarbassov, D. D. et al.,
Science 307, 1098-1101 (2005)). To determine whether Mfge8-induced
AKT phosphorylation was mediated through mTORC2, we evaluated
phosphorylation of rapamycin-insensitive companion of mTOR (Rictor)
at threonine 1135. Rictor was phosphorylated by Mfge8 and
phosphorylation required an intact integrin binding motif and at
least one discoidin domain (FIG. 4C). Mfge8 also induced
phosphorylation of AKT and Rictor in HepG2 cells by an
integrin-dependent pathway (FIGS. 4E,F).
Mfge8 Stimulates Fatty Acid Uptake by Inducing Translocation of
CD36 and FATP1 to the Cell Surface.
[0110] CD36 is a key mediator of fatty acid uptake (Ibrahimi, A. et
al., Proc Natl Acad Sci USA 93, 2646-2651 (1996)) that is regulated
by translocation from cytosolic stores to the cell surface (Glatz,
J. F. et al., Physiol Rev 90, 367-417 (2010)). We therefore
evaluated whether Mfge8 induced translocation of CD36 to the cell
surface. In primary Mfge8-/- adipocytes, hepatocytes, and
enterocytes there was a marked reduction in membrane CD36 (FIGS.
5A-C). The addition of rMfge8 increased membrane expression of CD36
in Mfge8-/- cells to WT levels, and this effect was completely
inhibited by wortmannin. Incubation with a CD36 blocking antibody
prevented rMfge8 from increasing fatty acid uptake in Mfge8-/-
adipocytes, hepatocytes, and enterocytes (FIGS. 5D-F). Unlike WT
adipocytes, rMfge8 did not significantly increase fatty acid uptake
in CD36-/- adipocytes. FATP1 is a member of the FATP family and
plays a key role in fatty acid transport in adipocytes. Like CD36,
FATP1 is translocated from cytosolic pool to the cell surface for
fatty acid transport (Stahl, A. et al., Developmental cell 2,
477-488 (2002)). Membrane expression of FATP1 was reduced in
Mfge8-/- adipocytes, expression was increased by rMfge8, and this
effect was inhibited by wortmannin (FIG. 5H). The addition of
rMfge8 to FATP1-/- adipocytes did not significantly increase fatty
acid uptake (FIG. 5I). Taken together, these data indicate that
Mfge8 regulates fatty acid uptake by inducing translocation of the
machinery of fatty acid uptake to the cell surface.
Mfge8-/- Mice are Protected from Weight Gain on a HFD Due to
Impaired Fat Absorption.
[0111] To determine whether Mfge8-mediated fatty acid uptake
contributes to the development of obesity in vivo, we evaluated
weight gain in Mfge8-/- mice placed on a HFD. Male and female
Mfge8-/- mice gained less weight as compared with controls over a
12 week period on a HFD. The eWAT of 20-week-old Mfge8-/- mice on a
HFD weighed significantly less than control eWAT. There was a
marked induction of Mfge8 protein in eWAT of WT mice on HFD.
20-week-old Mfge8-/- mice on a HFD or CD had smaller adipocytes and
reduced hepatic triglyceride content (CD). The hearts of Mfge8-/-
mice also had significantly reduced triglyceride content. To
determine the body composition of Mfge8-/- mice on a HFD, we
examined lean and fat mass using DEXA scanning. 20-week old
Mfge8-/- mice on HFD and 10- and 20-week old, but not 5-week-old,
Mfge8-/- mice on a CD had significantly less total body fat and
percent body fat. Since obesity is associated with insulin
resistance, we performed insulin tolerance tests on Mfge8-/- mice.
20-week old Mfge8-/- mice on a HFD had increased insulin
sensitivity compared to WT mice. 10-, but not 5-, week old Mfge8-/-
mice on a CD had enhanced insulin sensitivity. To evaluate whether
the decrease in body fat was secondary to impaired absorption of
dietary fats, we measured stool triglyceride and energy content.
Mfge8-/- mice on HFD had significantly higher stool triglyceride
levels and caloric content as measured by bomb calorimetry.
Mfge8-/- Mice are Protected from Obesity-Induced Adipose Tissue
Inflammation
[0112] Since obesity is associated with chronic inflammation
(Weisberg, S. P. et al., J Clin Invest 112, 1796-1808 (2003)) and
Mfge8 suppresses inflammation by multiple mechanisms (Kudo, M. et
al., Proc Natl Acad Sci USA 110, 660-665 (2013)), we evaluated eWAT
inflammation in Mfge8-/- mice on a HFD. There was a marked
reduction in eWAT infiltrating macrophages by immunohistochemistry
as well as a reduction in multiple immune populations as evaluated
by flow cytometry in Mfge8-/- mice. Since Mfge8-/- mice develop
age-dependent autoimmune disease (that is apparent at 40 weeks of
age) (Hanayama, R. et al., Science 304, 1147-1150 (2004)), we
evaluated whether 20-week old Mfge8-/- mice on a HFD had evidence
of chronic immune activation that may have contributed to the
decrease in body fat. We found no difference in the number or
percent of activated splenic lymphocytes or total number of cells
in the spleens of 20-week old Mfge8-/- mice on a HFD.
Mfge8 Deficiency does not Alter Energy Expenditure
[0113] We next examined whether differences in energy expenditure
could account for the differences in body fat in Mfge8-/- mice.
After 10 days on HFD, we placed 12-week-old Mfge8-/- and WT mice in
clams metabolic cages for a period of 4 days. We found no
difference in total oxygen consumption, oxygen consumption
corrected for lean body mass, food intake, or ambulation when
comparing Mfge8-/- and control mice. Finally, there was a modest
increase in the respiratory exchange ratio in Mfge8-/- mice without
changes in eWAT PGC1a expression. Collectively, these data indicate
that the decrease in body fat in Mfge8-/- mice is not caused by
exaggerated inflammation, an increase in energy expenditure, or an
increase in fatty acid oxidation.
Discussion
[0114] The work presented here identifies a critical role for Mfge8
in regulating obesity through modulation of cellular uptake and
storage of fatty acids. In the gastrointestinal tract, Mfge8
coordinates orderly absorption of dietary fats. Mfge8 deficiency
leads to fat malabsorption, a reduction in total body fat, and
protection from diet induced obesity (DIO). Importantly, Mfge8 also
increases fatty acid uptake by adipocytes, cardiac myocytes and
hepatocytes. While the in vivo sequelae of impaired fatty acid
uptake by peripheral organs in Mfge8-/- mice is obscured by the
malabsorption phenotype, the data suggests that under normal
conditions (wild type mice and humans), Mfge8 is physiologically
important for fat uptake in these tissues. This conclusion is
supported by the following observations. Serum fatty acid levels
are lower in Mfge8-/- mice after an olive oil bolus. However, when
mice are fasted for 24 hours, serum fatty acid levels are
significantly higher in Mfge8-/- mice consistent with impaired
peripheral uptake of fatty acids released after
catecholamine-induced lipolysis. There is a near 100-fold increase
in adipose tissue Mfge8 expression when mice are placed on a HFD
suggesting, as further discussed below, a key role for Mfge8 in
persistent expansion of adipose tissue with progressive weight
gain. Similarly, expression of Mfge8 and both the .alpha.v and
.beta.5 integrin subunits are significantly increased in the
adipose tissue of obese humans (Henegar, C. et al., Genome Biol 9,
R14 (2008)). Mfge8 induces translocation of FATP1 to the cell
surface, a fatty acid transport protein that is lacking in the
intestine, but present in adipocytes and cardiac myocytes.
[0115] Fatty acid transporter translocation is a key regulatory
step by which cellular uptake of fatty acids can be acutely
modified in response to hormonal and metabolic cues (Bonen, A. et
al., J Biol Chem 275, 14501-14508 (2000)). Insulin and muscle
contraction increase fatty acid uptake in skeletal and cardiac
muscle through this mechanism (Jain, S. S. et al., FEBS Lett 583,
2294-2300 (2009); Glatz, J. F. et al., Physiol Rev 90, 367-417
(2010)). The identification of additional regulatory pathways that
modulate this process has been elusive. While both insulin and
Mfge8 induce translocation of fatty acid transporters through PI3
kinase-dependent phosphorylation of AKT (Stahl, A. et al.,
Developmental cell 2, 477-488 (2002); Chabowski, A. et al., Am J
Physiol Endocrinol Metab 287, E781-789 (2004)), there are some key
differences between their roles in fatty acid uptake. In the
gastrointestinal tract, Mfge8 mediates absorption of dietary
triglycerides while insulin regulates nutrient deposition in
tissues after absorption from the intestine. Another distinction is
apparent in obese adipose tissue. While insulin promotes
lipogenesis by inducing de novo triglyceride synthesis (Wakil, S.
J. et al., Annu Rev Biochem 52, 537-579 (1983)), increasing fatty
acid uptake (Stahl, A. et al., Developmental cell 2, 477-488
(2002)), and inhibiting lipolysis (Jensen, M. D. et al., Diabetes
38, 1595-1601 (1989)), obesity is associated with adipocyte insulin
resistance (Smith, U., Int J Obes Relat Metab Disord 26, 897-904
(2002)). Adipocytes from diabetic patients have impaired downstream
signaling after insulin receptor binding, resulting in reduced PI3
kinase activity (Rondinone, C. M. et al., Proc Natl Acad Sci USA
94, 4171-4175 (1997)). This raises an interesting paradox: how does
insulin continue to promote adipocyte enlargement and obesity by
stimulating cells that are known to be insulin-resistant? Our data
raise the interesting possibility that the marked increase in
adipocyte Mfge8 expression during obesity may supply the signal
that promotes fatty acid uptake in insulin-resistant adipocytes,
thereby perpetuating the adipose hypertrophy characteristic of
obesity. Finally, unlike insulin, Mfge8 systemically induces fatty
acid uptake without directly affecting glucose uptake, thereby
providing a mechanism to dissociate regulation of these two major
components of nutrient metabolism.
[0116] The relative contribution of Mfge8-mediated CD36
translocation to increased fatty acid uptake induced by Mfge8 in
different organ system is an area of active investigation. In
adipocytes, our data indicate that Mfge8 regulates both FATP1 and
CD36 translocation. The roles of CD36 and FATP1 in promoting fatty
acid uptake are well established in adipocytes (Coburn, C. T. et
al., J Biol Chem 275, 32523-32529 (2000); Wu, Q. et al., Mol Cell
Biol 26, 3455-3467 (2006)), and cardiac myocytes (Coburn, C. T. et
al., J Biol Chem 275, 32523-32529 (2000); Tanaka, T. et al., J
Lipid Res 42, 751-759 (2001)) and are consistent with a model
whereby the effect of Mfge8 on fatty acid uptake in these tissues
is mediated through translocation of FATP1 and CD36 to the cell
surface. Whether the effect of Mfge8 on fatty acid uptake in the
intestinal tract is primarily mediated through CD36 is less clear.
Absorption of dietary fats is a multistep process that begins with
luminal breakdown of ingested triglycerides into free fatty acids
that are subsequently taken up by enterocytes where they are
re-esterified and secreted as chylomicrons (Bamba, V. & Rader,
D. J., Gastroenterology 132, 2181-2190 (2007)). CD36 modulates both
absorption of dietary fats and secretion of triglycerides by
intestinal epithelial cells (Drover, V. A. et al., J Biol Chem 283,
13108-13115 (2008); Drover, V. A. et al., J Clin Invest 115,
1290-1297 (2005); Nassir, F. et al., J Biol Chem 282, 19493-19501
(2007)). The impairment in Mfge8-/- enterocyte fatty acid uptake in
vitro and the increase in fecal energy content in Mfge8-/- mice
suggest that the main effect of Mfge8 is to stimulate uptake of
fatty acids rather than regulate secretion of chylomicrons. As we
found in adipocytes, Mfge8 may interact with additional fatty acid
transporters in the gastrointestinal tract leading to overlapping
but not identical phenotypes in enteral fat absorption in Mfge8-/-
and CD36-/- mice. While the protection from the DIO in Mfge8-/-
mice is reminiscent of MGAT2-/- mice, MGAT2-/- mice have a delay in
absorption rather than malabsorption of dietary fats. Yen, C. L. et
al., Nat Med 15, 442-446 (2009)).
[0117] Our work also identifies an important role for integrins in
regulating lipid homeostasis. We show that both the .alpha.v.beta.3
and .alpha.v.beta.5 integrins induce AKT phosphorylation via PI3
kinase and mTORC2. Of note, integrins are overexpressed in many
malignancies (Mizejewski, G. J., Proc Soc Exp Biol Med 222, 124-138
(1999)) and overexpression is important in the interaction of
malignant cells with the extracellular matrix relative to both
cancer growth and metastasis (Zhao, Y. et al., Cancer Res 67,
5821-5830 (2007)). Our data raise the possibility that integrin
overexpression in malignancies may increase tumor cell fatty acid
uptake. This may be particular importance in malignancies such as
prostate cancer where cells preferentially metabolize fatty acids.
(Liu, Y., Anticancer Res 30, 369-374 (2010)) and overexpress the
.alpha.v.beta.3 integrin (Zheng, D. Q., Cancer Res 59, 1655-1664
(1999)).
[0118] Our results provide a mechanism to explain the recent
observations that Mfge8 is located in a region linked with
susceptibility to obesity in humans (Rankinen, T. et al., Obesity
(Silver Spring) 14, 529-644 (2006)) and that adipose expression of
Mfge8 is increased in human obesity (Henegar, C. et al., Genome
Biol 9, R14 (2008)). Collectively, our data indicate that Mfge8
ligation of integrin receptors regulates body fat content by
regulating the uptake of fatty acids in the alimentary tract and in
peripheral tissues. From the therapeutic viewpoint, this pathway
can be targeted for the treatment of malabsorption syndromes or
obesity. To our knowledge, we show the first evidence of a pathway
that can augment absorption of dietary fats and serve as a target
for the treatment of fat malabsorption. In addition, inhibition of
the Mfge8-dependent pathway will provide a novel therapeutic target
for the treatment of obesity that directly inhibits the molecular
pathways of fat absorption in the gastrointestinal tract. A better
understanding of the mechanisms that regulate fat uptake and
storage is of significant interest in the light of the high
morbidity, mortality and economic burden associated with obesity
and obesity-related disease.
Supplemental Methods
[0119] Mice.
[0120] All animal experiments were approved by the UCSF
Institutional Animal Care and Use Committee in adherence to NIH
guidelines and policies. In vivo studies were conducted with two
different lines of mice deficient in Mfge8. Some studies were
carried out on Mfge8-/- mice created by a gene disruption vector.
These mice have been extensively characterized and have the same
phenotypes as Mfge8-/- mice created by homologous recombination
(Silvestre, J. S. et al., Nat Med 11, 499-506 (2005); Atabai, K. et
al., Mol Biol Cell 16, 5528-5537 (2005)). Mice were backcrossed 10
generations into the C57bl/6 background and bred as Mfge8-/-
breeding pairs and Mfge8+/+ breeding pairs. In a subset of studies,
Mfge8-/- and Mfge8+/- breeding pairs were used to generate sibling
littermates from the same cage. A second line of Mfge8 mice created
by homologous recombination was obtained from RIKEN (Hanayama, R.
et al., Nature 417, 182-187 (2002)). These mice were bred as
Mfge8-/- and Mfge8+/- breeding pairs and used in some studies and
as Mfge8-/- and Mfge8+/+ breeding pairs for studies used in FIGS.
3A-3K and, and for harvesting of all primary cells used in in vitro
studies. All mice were age- (6-8 weeks of age unless otherwise
noted) and sex-matched. .beta.3-/- and .beta.5-/- mice in the 129
SVEV strain have been previously described (Huang, X. et al., Mol
Cell Biol 20, 755-759 (2000); Su, G. et al., Am J Respir Crit Care
Med 185, 58-66 (2012)). CD36-/- mice were generously provided by
Roy Silverstein and were in the C57bl/6 background. FAP1-/- mice
were also in the C57bl/6 background (Wu, Q. et al., Mol Cell Biol
26, 3455-3467 (2006)). For FIGS. 3C-3I investigator were blinded to
genotypes until statistical analysis of the data. Investigators
were not blinded as to genotype in animal studies that involved
weighing mice on a high-fat diet, obtaining insulin tolerance
tests, and determining body composition by Dexa scan. Investigators
were blinded to the mouse genotypes for the energy expenditure
experiments which were done by a core facility.
[0121] High-Fat Diet.
[0122] 8 to 10 week-old mice were placed on a high-fat formula
containing 60% fat calories (Research Diets, Inc.) for 12 weeks.
The control diet contained 9% fat calories (PMI). Mice were housed
in groups of 5 mice per cage for diet experiments including
weights, insulin tolerance tests, Dexa scanning for body
composition, adipocyte size quantification, and hepatic
triglyceride content with each cage of 5 mice representing an
independent experiment.
[0123] Fluorescent Fatty Acid Uptake Assay.
[0124] Uptake of fatty acids by differentiated 3T3-L1 adipocytes or
primary mouse adipocytes was assessed using a QBT Fatty Acid Uptake
Kit (Molecular Devices). Cells were plated in triplicate in 96-well
plates at a concentration of 25,000 cells per well in 100 .mu.l of
DMEM/10% FCS. Plates were centrifuged at 1000 rpm for 4 minutes and
incubated at 37.degree. Celsius for 4-5 hours. Cells were then
serum deprived for 1 hour before treatment with recombinant
proteins for 30 minutes followed by the addition of QBT Fatty Acid
Uptake solution. In experiments using function-blocking antibodies,
antibodies against mouse integrins .alpha.v (clone RMV-7)
(Takahashi, K. et al., J Immunol 145, 4371-4379 (1990)).beta.3
(clone 2C9.G2; BD Biosciences) (Ashkar, S. et al., Science 287,
860-864 (2000)), .beta.5 (clone ALULA) (Su, G. et al., American
journal of respiratory cell and molecular biology 36, 377-386
(2007)), .beta.1 (clone HA2/5; BD Biosciences, anti-rat with
cross-reactivity with mouse (Zovein, A. C. et al., Developmental
cell 18, 39-51 (2010)), CD36 (clone MF3; Abcam) (Helming, L. et
al., Journal of cell science 122, 453-459 (2009)), human integrins
.alpha.v (clone L230) (Thomas, G. J. et al., British journal of
cancer 87, 859-867 (2002)), .beta.3 (clone Axum-2) (Su, G. et al.,
Am J Respir Crit Care Med 185, 58-66 (2012)), .beta.5 (clone
ALULA), .beta.1 (clone P5D2), cycloRGD and cycloRAD (BACHEM) were
added to cells after serum deprivation and cells were incubated for
20 minutes at 4.degree. Celsius prior to addition of recombinant
proteins. Plates were incubated in a fluorescent plate reader at
37.degree. Celsius and kinetic readings were acquired every 20
seconds for 30 minutes. Fluorescence values were plotted against
time and data was expressed as relative fluorescent units per
minutes.times.10.sup.3.
[0125] 3T3-L1 Cell/HepG2 Cell Culture.
[0126] 3T3-L1 (Zen-Bio) fibroblasts were differentiated into
adipocytes as described previously (Liao, J. et al., J Lipid Res
46, 597-602 (2005)). Briefly, 3T3-L1 fibroblasts were cultured to
confluence on 10 cm tissue culture plates or in 6-well tissue
culture dishes in DMEM supplemented with 10% FBS and 25 mM HEPES
(normal medium). 2 days after reaching confluence, media was change
and 3-isobutyl-1methylxanthine (Calbiochem), dexamethasone (Sigma),
and insulin (Sigma) were added to the normal medium at
concentrations of 0.5 mM, 1 .mu.M, and 5 .mu.g/mL, respectively, to
induce adipocyte differentiation. After 2 days (and every 2 days
thereafter), media was replaced with normal medium supplemented
with 5 .mu.g/mL insulin. Cells were harvested for use 6-10 days
after differentiation. The human hepatocellular carcinoma cell line
HepG2 was a generous gift of Dr. Ethan Weiss. Cells were propagated
in Eagle's MEM supplemented with 10% fetal bovine serum.
[0127] Olive Oil Gavage and Serum Triglyceride and Fatty Acid
Content.
[0128] 6-8 week-old mice were fasted for 4 hours and then gavaged
with 15 .mu.L olive oil per gram body weight. Mice had access to
water but not food for the remainder of the experiment. In the
experiments in FIGS. 3C and 3F, 50 .mu.g/kg body weight of
recombinant protein was mixed into olive oil and administered
immediately to mice by gavage. In blocking antibody experiments,
integrins .alpha.v (clone RMV-7) and .beta.5 (clone ALULA) were
administered by gavaging mice with 100 .mu.L water containing 0.5
.mu.g antibody per gram body weight 30 minutes before olive oil
gavage. Triglyceride content of serum was assayed using a
commercially available kit (Sigma-Aldrich) and fatty acid
concentrations were quantified by Wako (Rabot, S. et al., FASEB J
24, 4948-4959 (2010)).
[0129] Quantification of Liver, Intestinal, and Fecal Triglyceride
Content.
[0130] After experiments described above, samples from the left
lobe of the liver and the proximal small intestine were isolated
and rapidly frozen in liquid nitrogen for triglyceride content
assays. Control mice were starved for 8 hours before their organs
were harvested as above. Triglyceride content of the intestine
(Uchida, A. et al., Front Physiol 3, 26) and liver (Kim, K. Y. et
al., J Clin Invest 121, 3701-3712 (2011)) and fecal samples Kim, K.
Y. et al., J Clin Invest 121, 3701-3712 (2011)) were quantified as
described previously.
[0131] Glucose Gavage and Blood Glucose Measurement.
[0132] 6-8 week-old mice were fasted for 4 hours and then gavaged
with 1.5 mg glucose per gram body weight. In the experiments in
FIG. 7A, 50 .mu.g/kg body weight of recombinant protein was mixed
into glucose solution and administered immediately to mice by
gavage. In blocking antibody experiments, integrins .alpha.v (clone
RMV-7) and .beta.5 (clone ALULA) were administered orally with 100
.mu.L water containing 0.5 .mu.g antibody per gram body weight 30
minutes before glucose gavage. Blood glucose levels were measured
by sampling from the tail vein of mice from 0-60 minutes after
glucose administered.
[0133] Fecal Fatty Acid Content.
[0134] 6-8 week-old mice were fasted for 4 hours and then gavaged
with 2 .mu.g BODIPY per gram body weight. Feces were collected from
20 minutes to 4 hours after BODIPY administered. 50 mg of feces was
homogenized in PBS contained 30 mM HEPES, 57.51 mM MgCl.sub.2 and
0.57 mg/ml BSA with 0.5% SDS and sonicated for 30 seconds, and then
centrifuged at 1000 g for 10 minutes. Supernatants were transferred
to 96 well plates and Fluorescence values were measured immediately
using a fluorescence microplate reader for endpoint reading
(Molecular Devices).
Primary Adipocyte, Hepatocyte, Enterocyte, and Adipocyte Progenitor
Culture.
[0135] Adipocytes.
[0136] Primary mouse adipocytes were obtained from epididymal fat
pads by collagenase digestion in Krebs-HEPES (KRBH) buffer followed
by filtering through a 100 .mu.m strainer which was then washed
with an additional 7.5 mL KRBH buffer. Adipocytes were allowed to
float to the top of the mixture for 5 minutes, and the solution
under the adipocyte layer was removed with a syringe. The
adipocytes were washed with 10 mL KRBH and again allowed to float
to the surface, at which point the solution was again removed. This
process was repeated for a total of 3 washes. After the last
aspiration, adipocytes were resuspended in 0.5-1.0 mL and
counted.
[0137] Adipocyte Progenitors.
[0138] Primary mouse adipocyte progenitors from the vascular
stromal fraction were isolated and cultured as reported previously
(Tseng et al., 2008). In brief, subcutaneous white adipose tissue
was removed, minced and digested with 1 mg/ml collagenase for 45
min at 37.degree. C. in DMEM/F12 medium containing 1% BSA and
antibiotics. Digested tissues were filtered through sterile 150
.mu.m nylon mesh and centrifuged at 250 g for 5 min. The floating
fractions consisting of adipocytes were discarded and the pellets
representing the stromal vascular fractions were resuspended in
erythrocyte lysis buffer (154 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1
mM EDTA) for 10 min to remove red blood cells. The cells were
further centrifuged at 500 g for 5 min, plated at 8.times.10.sup.5
per well of a 24-well plate, and grown at 37.degree. C. in DMEM/F12
supplemented with 10% FBS at 37.degree. C. 2 days after cells
reached 100% confluence, cells were treated with 1 .mu.M
rosiglitazone and 5 .mu.g/mL insulin to induce terminal
differentiation.
[0139] Primary Hepatocytes.
[0140] The liver was pre-perfused through the portal vein with
calcium-free buffer (0.5 mM EDTA, HBSS without Ca.sup.2+ and
Mg.sup.2+) and next perfused with collagenase (3.5 U/ml Collegenase
II (Worthington) 25 mM HEPES, HBSS with Ca.sup.2+ and Mg.sup.2+).
Parenchymal cells were purified by Percoll buffer (90% Percoll
(Sigma), 1.times.PBS) at low-speed centrifugation (1500 rpm for 10
min). Viability of isolated hepatocytes was determined by Trypan
blue staining (around 85%) and cell density was approximately 60%
confluence. Cells were plated in collagen-I-coated dishes and
cultured at 37.degree. C. in a humidified atmosphere of 95% O.sub.2
and 5% CO.sub.2 in growth medium (Huang, P. et al., Nature 475,
386-389 (2011)).
[0141] Primary Enterocytes.
[0142] The proximal small intestine was collected from anesthetized
mice, and the luminal contents were emptied, washed with 115 mM
NaCl, 5.4 mM KCl, 0.96 mM NaH.sub.2PO.sub.4, 26.19 mM NaHCO.sub.3,
and 5.5 mM glucose buffer, pH 7.4, and gassed for 30 minutes with
95% O.sub.2 and 5% CO.sub.2. The proximal small intestines were
then filled with buffer containing 67.5 mM NaCl, 1.5 mM KCl, 0.96
mM NaH.sub.2PO.sub.4, 26.19 mM NaHCO.sub.3, 27 mM sodium citrate,
and 5.5 mM glucose, pH 7.4, saturated with 95% O.sub.2 and 5%
CO.sub.2, and incubated in a bath containing oxygenated saline at
37.degree. C. with constant shaking. After 15 minutes, the luminal
solutions were discarded and the intestines were filled with buffer
containing 115 mM NaCl, 5.4 mM KCl, 0.96 mM NaH.sub.2PO.sub.4,
26.19 mM NaHCO.sub.3, 1.5 mM EDTA, 0.5 mM dithiothreitol, and 5.5
mM glucose, pH 7.4, saturated with 95% O.sub.2 and 5% CO.sub.2, and
bathed in saline as described above. After 15 minutes, the luminal
contents were collected and centrifuged (1,500 rpm, 5 minutes, room
temperature), and the pellets were resuspended in DMEM saturated
with 95% O.sub.2 and 5% CO.sub.2.dbd. (Anwar, K. et al., J Lipid
Res 48, 2028-2038 (2007)).
[0143] Primary Cardiomyocytes.
[0144] Hearts were immersed in ice-cold calcium-free perfusion
buffer containing (in mmol/L) NaCl 120.4, KCl 14.7,
KH.sub.2PO.sub.4 0.6, Na.sub.2HPO.sub.4 0.6, 5 MgSO.sub.4-7H.sub.2O
1.2, Na-HEPES 10, NaHCO.sub.34.6, taurine 30, butanedione monoxime
(BDM) 10, glucose 5.5, and then perfused through the aorta with
calcium-free perfusion buffer (3 ml/minutes) for 4 minutes, then
switched to calcium-free digestion buffer (perfusion buffer
containing collagenase II [2 mg/ml] from Worthington Biochemical)
for 10 minutes. This was followed by perfusion with digestion
buffer containing 100 .mu.mol/L CaCl.sub.2) for another 8-10
minutes. Hearts were removed from the perfusion apparatus and
placed in a 10 cm Petri dish containing 2 ml digestion buffer and 3
ml of stop buffer (perfusion buffer supplemented with 10% FBS). The
atria were removed and the ventricles were pulled into 10-12
equally sized pieces. Tissue was then gently dispersed into cell
suspension using plastic transfer pipettes. The cell suspension was
collected in a 15 ml falcon tube, brought to 10 ml with stop buffer
and centrifuged at 40.times.g for 3 minutes. Damaged myocytes and
non-myocytes were removed by a series of washes in 10 ml stop
buffer containing, sequentially, 100, 400, or 900 .mu.mol/L
CaCl.sub.2). Cardiomyocytes were pelleted by centrifugation at
40.times.g for 3 minutes after each wash and plated in laminin
coated dishes (Smyth, J. W. et al., Circ Res 110, 978-989
(2012)).
[0145] Recombinant Protein Production.
[0146] Recombinant protein constructs were created and expressed in
High 5 cells as previously described (Atabai, K. et al., J Clin
Invest 119, 3713-3722 (2009)). For studies using different
recombinant constructs the molar equivalent of 10 .mu.g/mL of
full-length recombinant Mfge8 was used for each construct.
[0147] Western Blot.
[0148] Following tissue preparation and SDS-PAGE, membranes were
incubated with a polyclonal antibody against Akt (Cell Signaling
Technology), or Rictor (Cell Signaling); Anti-PGC1 alpha antibody
(abcam); a monoclonal antibody against Phospho-Akt Ser473 (Cell
Signaling), Phospho-Rictor Thr1135 (Cell Signaling), GAPDH (Cell
Signaling), or Mfge8 (R&D Systems). For evaluation of total AKT
and total Rictor, membranes that been blotted for phospho-AKT and
phospho-Rictor were stripped and reprobed. Plasma membrane and
post-plasma membrane fractions were isolated as previously
described (Nishiumi, S. & Ashida, H., Biosci Biotechnol Biochem
71, 2343-2346 (2007)).
[0149] Immunohistochemistry.
[0150] 5 .mu.m sections were boiled for 15 minutes in 10 mM sodium
citrate (pH 6) for antigen retrieval and blocked with
H.sub.2O.sub.2 in methanol and subsequently 2% BSA. Rabbit
anti-MAC2 antibody (Cedarlane, CL8942AP) directed against MAC2 was
used at 1:3800 dilution in TBS and 0.5% tween, followed by a 1:200
biotinylated anti-rabbit secondary antibody (Vector), ABC reagent
(Vector) and liquid diaminobenzidine substrate (Sigma).
[0151] Morphometric Analysis.
[0152] Paraffin embedded eWAT sections from 5 CD and 10 HFD mice
were stained with H&E. For each section 5 high-power field
(HPF) pictures were taken at 100.times. magnification. The average
number of adipocytes per HPF for each section was counted and the
diameter of each adipocyte was measured using Image-Pro Plus MDA.
Investigators were blinded to genotype during quantification.
[0153] Flow Cytometry.
[0154] Epididymal fat pads were dissected, weighed, and placed in a
buffered collagenase solution for homogenization using a GentleMACS
tissue dissociator. Homogenized tissue was incubated at 37.degree.
C. on a rotating shaker at 250 rpm for 30 minutes, then passed
through a 40 um strainer and rinsed with 10 mL ice-cold PBS. After
a red blood cell lysis step, cells were stained for viability using
a LIVE/DEAD aqua fixable stain kit (Invitrogen, Carlsbad, Calif.)
and then for the following stains to identify macrophage subtype
and eosinophil populations: CD45 (clone 30-F11, BioLegend, San
Diego, Calif.), CD11b (clone M1/70, BioLegend), F4/80 (clone BM8,
BioLegend), CD11c (clone N418, BioLegend), CD301 (clone ER-MP23,
AbdSerotec, Oxford, United Kingdom), Siglec (clone E50-2440, BD
Pharmingen, San Diego, Calif.). A second set of cells from the fat
pads were similarly stained for viability and then with the
following antibodies to identify lymphocyte populations: CD45, CD4
(clone RM4-4, BioLegend), CD44 (clone IM7, Ebioscience), CD62L
(clone MEL-14, BD Pharmingen), and FoxP3 (clone FJK-16s,
Ebioscience, San Diego, Calif.). Spleens were removed after
sacrificing ironic within. The splenocytes were treated to lyse red
blood cells and subsequently stained for viability and the
lymphocyte markers detailed above. Flow cytometry was performed on
a BD FACS flow cytometer and analyzed using FlowJo Software (Tree
Star Inc., Ashland, Oreg.).
[0155] Body Fat Analysis.
[0156] Bone, lean, and fat mass analysis was performed with GE
Lunar PIXImus II Dual Energy X-ray Absorptiometer.
[0157] CLAMS Metabolic Cage Analysis.
[0158] Mice were placed in single housing cages for 5 days prior to
initiating experimental analysis for a period of 96 hours. Mice
were on a HFD for 10 days prior to initiating the analysis. The
following variables were measured: food and water intake, oxygen
consumption (VO.sub.2) and carbon dioxide production (VCO.sub.2)
(at 13 minute intervals), and locomotor activity. Infrared beams
monitored movement in the X, Y, and Z directions. The data
presented was from the last 48 hours of the analysis (Sutton, G. M.
et al., Endocrinology 147, 2183-2196 (2006)).
[0159] Measurements of Fecal Energy Content.
[0160] Feces from mice on a HFD were freeze dried (samples from 2
mice were combined for each sample) and pulverized with a ceramic
mortar and pestle. Caloric content of feces was measured with an
1108 Oxygen Combustion Bomb calorimeter.
[0161] Insulin Tolerance Tests.
[0162] For insulin tolerance test, mice were fasted for 5 hours
after which they were injected with 1.5 U/kg of insulin IP. Blood
was collected from the tail vein immediately before injection and
then again after 15, 30, 60, and 90 minutes for evaluation of blood
glucose.
Statistical Analysis
[0163] Data were assessed for normal distribution and similar
variance between groups using Graphpad Prism 6.0. One-way ANOVA was
used to make comparisons between multiple groups. When the ANOVA
comparison was statistically significant (P<0.05), further
pairwise analysis was performed using a Bonferroni t-test. 2-sided
Student's t-test, Mann-Whitney t-test or unpaired t-test with
Welch's correction was used for comparisons between 2 groups
depending on the distribution and variance of the data. GraphPad
Prism 6.0 was used for all statistical analysis. All data are
presented as mean.+-.s.e.m. Sample size for animal experiments were
selected based on numbers typically used in the literature. There
was no randomization of animals.
Example 2: Mfge8 Modulates Glucose Uptake In Vitro and In Vivo
[0164] This example illustrates that the administration of
recombinant Mfge8 (rMfge8) induces acute insulin resistance in
cultured adipocytes and in vivo, while the administration of
.alpha.v or .beta.5 integrin blocking antibodies significantly
increased the effect of insulin on glucose uptake in vitro and in
vivo. This example also shows that IP co-administration of rMfge8
and insulin in mice resulted in significantly higher serum glucose
levels as compared with insulin injection alone. Furthermore,
pretreatment with .alpha.v or .beta.5 integrin blocking antibody
prior to insulin injection resulted in significantly lower serum
glucose levels after insulin injection as compared to insulin
injection alone.
[0165] FIG. 9 shows that Mfge8 induces insulin resistance in 3T3-L1
adipocytes. Glucose uptake in 3T3-L1 adipocyte with and without 20
min treatment with recombinant Mfge8 or RGE (10 .mu.g/ml) and
insulin (1 .mu.M) or both mfge8 and insulin (n=8, P<0.05). Data
are expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment.
[0166] FIG. 10 shows that integrin receptor blockade enhances
insulin sensitivity in primary adipocytes. Glucose uptake in
Mfge8-/- and Mfge8+/+ primary adipocytes, with and without 20 min
treatment with insulin (1 .mu.M) and effect of pretreatment with
integrin blocking antibodies (0.5 .mu.g/g, IP, 15 min before
insulin) on glucose uptake in Mfge8+/+ adipocytes. Data are
expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment (n=8, P<0.05). Pretreatment with av, b3
or b5 integrin blocking antibody prior to insulin injection
resulted in significantly lower serum glucose levels after insulin
injection as compared with insulin injection alone.
[0167] FIG. 11 shows that Mfge8 induces acute insulin resistance in
vivo. 8-week-old Mfge8-/- and Mfge8+/+ control mice were fasted for
4 hours, then blood glucose was measured 15 min after IP injection
of insulin (1 U/kg), saline, RGE (50 .mu.g/kg) or a combination of
insulin (1.5 U/Kg) and rMfge8 or RGE construct (50n/kg). Data are
expressed as mean.+-.s.e.m. Each replicate represents an
independent experiment (n=4, P<0.05).
[0168] FIG. 12 shows that integrin blockade induces acute insulin
sensitivity in vivo. 8-week-old Mfge8-/- and Mfge8+/+ control mice
were fasted for 4 hours, then received blocking antibodies (0.5
.mu.g per gram body weight) (.alpha.v (clone RMV-7) and .beta.v
(clone ALULA)) IP. 15 min prior insulin (1 U/kg) or saline, blood
glucose was measured 15 min after IP injection of insulin (1 U/kg)
or saline. Data are expressed as mean.+-.s.e.m. Each replicate
represents an independent experiment (n=4, P<0.05).
[0169] While recombinant Mfge8 (rMfge8) had no effect on baseline
glucose uptake, rMfge8 significantly inhibited while .alpha.v or
.beta.5 integrin blocking antibodies significantly increased the
effect of insulin on glucose uptake in both 3T3-L1 adipocytes and
primary WT adipocytes. IP co-administration of rMfge8 and insulin
in mice resulted in significantly higher while pretreatment with
.alpha.v or .beta.5 integrin blocking antibody prior to insulin
injection resulted in significantly lower serum glucose levels
after insulin injection as compared with insulin injection alone.
These data indicate that Mfge8 acutely modulates the glucose uptake
response to insulin. Co-injection of .alpha.v or .beta.5 integrin
blocking antibodies with insulin may reduce insulin requirements by
approximately 50%. Systemic therapy with .alpha.v or .beta.5
integrin blocking antibodies may enhance insulin sensitivity in
at-risk populations of individuals.
[0170] Glucose Uptake Assay.
[0171] Uptake of fatty acids by primary cells and cell lines was
assessed using the fluorescent D-glucose analog
2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose
(2-NBDG) (Invitrogen). 2-NBDG has been successfully used as an
alternative to 2-deoxyglucose in the measurement of glucose uptake
in multiple cell types. Cells were plated in triplicate in 96-well
plates at a concentration of 25,000 cells per well in 100 .mu.l of
DMEM/10% FCS. Plates were centrifuged at 1000 rpm for 4 minutes and
incubated at 37.degree. C. for 4-5 hours. Cells were then serum
deprived for 1 hour before treatment with recombinant proteins for
20 minutes followed by the addition of 2-NBDG (100 .mu.M) for 10
min at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2.
Reaction was stopped by adding a twofold volume of ice-cold PBS and
the wells were washed again with ice-cold PBS three times. The
fluorescent signal before (autofluorescence) and after adding 100
.mu.M 2-NBDG was measured using fluorescent plate reader at
37.degree. C. (Molecular Device using the 485 nmex and 520 nmemiss
filter set). The net increase in fluorescence was normalized to the
lowest signal (0 cells/well). In experiments using
function-blocking antibodies, antibodies against mouse integrins
.alpha.v (clone RMV-7), .beta.3 (clone 2C9.G2; BD Biosciences),
.beta.5 (clone ALULA), .beta.1 (clone HA2/5; BD Biosciences),
anti-rat with cross-reactivity with mouse, were added to cells
after serum deprivation and cells were incubated for 20 minutes
prior to addition of recombinant proteins.
[0172] Insulin Tolerance Test.
[0173] Mice were fasted for 4 hours after which they were injected
with 1.5 U/kg of insulin IP. Blood was collected from the tail vein
immediately before injection and then again after 15, 30, 60, and
90 minutes for evaluation of blood glucose.
[0174] Blood Glucose Measurement.
[0175] In experiments using RGE or Mfge8 construct: mice received
50 .mu.g/kg body weight of recombinant protein. In experiments
using function-blocking antibodies: 6-8 week-old mice were fasted
for 4 hours and then each mouse received 0.5 .mu.g antibody
(integrins .alpha.v (clone RMV-7) and .beta.5 (clone ALULA)) per
gram body weight in total volume of 200 .mu.l, 15 minutes later
each mouse received an insulin injection (1.5 U/kg, intraperitoneal
injection (IP)). Blood glucose levels were measured by sampling
from the tail vein of mice from 15 minutes after insulin
administered.
Example: Mfge8 Modulates Glucose Uptake In Vitro and In Vivo
[0176] This example illustrates enhanced, antral smooth muscle
contraction in Mfge8-/- mice. It also shows that PI3K inhibition
prevented exaggerated Mfge8-/- antral ring contraction.
[0177] FIGS. 13A-13C show the force of antral smooth muscle ring
contraction with and without the addition of the rMfge8 or RGE
construct (FIG. 13A) or after in vivo induction of transgenic Mfge8
expression in Mfge8-/-sm+ mice in response to MCh (FIG. 13B). FIG.
13C shows the force of antral contraction with and without
epithelium (denuded). Mfge8-/- mice have enhanced gastric emptying
and more rapid small intestine transit time (SIT). FIGS. 13D and
13E show that gastric emptying was measured by the proportion of
phenol red remaining in the stomach 15 minutes after gavage.
N=7-10. FIGS. 13F and 13G shows small intestinal transit times
after gavage with Carmine dye with subsequent evaluation at 15
minutes of dye migration along the intestinal tract. N=5-10 in FIG.
13C and 3-5 in FIG. 13D. In FIGS. 13E and 13G, Mfge8-/-sm+ and
single transgenic controls were placed on doxycycline for 2 weeks
prior to the experiments to induce Mfge8 production in the smooth
muscle. *P<0.05, **P<0.01, ***P<0.001.
[0178] In FIG. 14A, the antral rings were treated for 15 min with
PI3K inhibitor wortmannin (Wort. 100 nm) followed by assessment of
contractile force in response to MCh. N=3-4. ***P<0.0001. FIG.
14B shows that Mfge8 reduces AKT phosphorylation. Western blot of
antral tissue were treated for 30 minutes with or without rMfge8.
FIG. 14C shows that wortmannin prevents RhoA activation. Western
blot of antrum were treated for 30 minutes with wortmannin (100
ng/ml) or Mfge8 and then with MCh for 15 minutes prior to
quantifying active RhoA using a GST pull-down. FIG. 14D shows that
Mfge8 modulates PTEN activity. PTEN activity assay measured the
conversion of PIP3 to PIP2 in freshly isolated antrum with and
without the rMfge8 or RGE construct (10 .mu.g/ml) n=3-5.
*P<0.05, **P<0.01, ***P<0.001.
[0179] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
71335PRTHomo sapienshuman mature milk fat globule epidermal growth
factor 8 (MFGE8), lactadherin isoform b, O-acetyl
disialoganglioside synthase (OAAcGD3S), breast epithelial antigen
BA46, medin, sperm associated antigen 10 (SPAG10), sperm surface
protein hP47 1Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala
Leu Leu Cys1 5 10 15 Ala Pro Ser Leu Leu Val Ala Leu Asp Ile Cys
Ser Lys Asn Pro Cys 20 25 30 His Asn Gly Gly Leu Cys Glu Glu Ile
Ser Gln Glu Val Arg Gly Asp 35 40 45 Val Phe Pro Ser Tyr Thr Cys
Thr Cys Leu Lys Gly Tyr Ala Gly Asn 50 55 60 His Cys Glu Thr Lys
Cys Val Glu Pro Leu Gly Leu Glu Asn Gly Asn65 70 75 80 Ile Ala Asn
Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu 85 90 95 Gly
Leu Gln His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly 100 105
110 Met Val Asn Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile
115 120 125 Gln Val Asn Leu Leu Arg Arg Met Trp Val Thr Gly Val Val
Thr Gln 130 135 140 Gly Ala Ser Arg Leu Ala Ser His Glu Tyr Leu Lys
Ala Phe Lys Val145 150 155 160 Ala Tyr Ser Leu Asn Gly His Glu Phe
Asp Phe Ile His Asp Val Asn 165 170 175 Lys Lys His Lys Glu Phe Val
Gly Asn Trp Asn Lys Asn Ala Val His 180 185 190 Val Asn Leu Phe Glu
Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr 195 200 205 Pro Thr Ser
Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly 210 215 220 Cys
Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly Leu Lys Asn Asn Ser225 230
235 240 Ile Pro Asp Lys Gln Ile Thr Ala Ser Ser Ser Tyr Lys Thr Trp
Gly 245 250 255 Leu His Leu Phe Ser Trp Asn Pro Ser Tyr Ala Arg Leu
Asp Lys Gln 260 265 270 Gly Asn Phe Asn Ala Trp Val Ala Gly Ser Tyr
Gly Asn Asp Gln Trp 275 280 285 Leu Gln Ile Phe Pro Gly Asn Trp Asp
Asn His Ser His Lys Lys Asn 290 295 300 Leu Phe Glu Thr Pro Ile Leu
Ala Arg Tyr Val Arg Ile Leu Pro Val305 310 315 320 Ala Trp His Asn
Arg Ile Ala Leu Arg Leu Glu Leu Leu Gly Cys 325 330 335 2387PRTHomo
sapienshuman milk fat globule epidermal growth factor 8 preprotein
(MFGE8), lactadherin isoform a preproprotein, O-acetyl
disialoganglioside synthase (OAAcGD3S), breast epithelial antigen
BA46, sperm surface protein hP47, SPAG10 2Met Pro Arg Pro Arg Leu
Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys1 5 10 15 Ala Pro Ser Leu
Leu Val Ala Leu Asp Ile Cys Ser Lys Asn Pro Cys 20 25 30 His Asn
Gly Gly Leu Cys Glu Glu Ile Ser Gln Glu Val Arg Gly Asp 35 40 45
Val Phe Pro Ser Tyr Thr Cys Thr Cys Leu Lys Gly Tyr Ala Gly Asn 50
55 60 His Cys Glu Thr Lys Cys Val Glu Pro Leu Gly Leu Glu Asn Gly
Asn65 70 75 80 Ile Ala Asn Ser Gln Ile Ala Ala Ser Ser Val Arg Val
Thr Phe Leu 85 90 95 Gly Leu Gln His Trp Val Pro Glu Leu Ala Arg
Leu Asn Arg Ala Gly 100 105 110 Met Val Asn Ala Trp Thr Pro Ser Ser
Asn Asp Asp Asn Pro Trp Ile 115 120 125 Gln Val Asn Leu Leu Arg Arg
Met Trp Val Thr Gly Val Val Thr Gln 130 135 140 Gly Ala Ser Arg Leu
Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val145 150 155 160 Ala Tyr
Ser Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn 165 170 175
Lys Lys His Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His 180
185 190 Val Asn Leu Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu
Tyr 195 200 205 Pro Thr Ser Cys His Thr Ala Cys Thr Leu Arg Phe Glu
Leu Leu Gly 210 215 220 Cys Glu Leu Asn Gly Cys Ala Asn Pro Leu Gly
Leu Lys Asn Asn Ser225 230 235 240 Ile Pro Asp Lys Gln Ile Thr Ala
Ser Ser Ser Tyr Lys Thr Trp Gly 245 250 255 Leu His Leu Phe Ser Trp
Asn Pro Ser Tyr Ala Arg Leu Asp Lys Gln 260 265 270 Gly Asn Phe Asn
Ala Trp Val Ala Gly Ser Tyr Gly Asn Asp Gln Trp 275 280 285 Leu Gln
Val Asp Leu Gly Ser Ser Lys Glu Val Thr Gly Ile Ile Thr 290 295 300
Gln Gly Ala Arg Asn Phe Gly Ser Val Gln Phe Val Ala Ser Tyr Lys305
310 315 320 Val Ala Tyr Ser Asn Asp Ser Ala Asn Trp Thr Glu Tyr Gln
Asp Pro 325 330 335 Arg Thr Gly Ser Ser Lys Ile Phe Pro Gly Asn Trp
Asp Asn His Ser 340 345 350 His Lys Lys Asn Leu Phe Glu Thr Pro Ile
Leu Ala Arg Tyr Val Arg 355 360 365 Ile Leu Pro Val Ala Trp His Asn
Arg Ile Ala Leu Arg Leu Glu Leu 370 375 380 Leu Gly Cys385
3463PRTMus musculusmouse milk fat globule epidermal growth factor 8
(Mfge8, MFG-E8) long form, lactadherin isoform 1 precursor, sperm
surface protein SP47 (MP47, P47), EGF/Factor VIII, Mfgm 3Met Gln
Val Ser Arg Val Leu Ala Ala Leu Cys Gly Met Leu Leu Cys1 5 10 15
Ala Ser Gly Leu Phe Ala Ala Ser Gly Asp Phe Cys Asp Ser Ser Leu 20
25 30 Cys Leu Asn Gly Gly Thr Cys Leu Thr Gly Gln Asp Asn Asp Ile
Tyr 35 40 45 Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Val Cys Asn
Glu Thr Glu 50 55 60 Arg Gly Pro Cys Ser Pro Asn Pro Cys Tyr Asn
Asp Ala Lys Cys Leu65 70 75 80 Val Thr Leu Asp Thr Gln Arg Gly Asp
Ile Phe Thr Glu Tyr Ile Cys 85 90 95 Gln Cys Pro Val Gly Tyr Ser
Gly Ile His Cys Glu Thr Glu Thr Asn 100 105 110 Tyr Tyr Asn Leu Asp
Gly Glu Tyr Met Phe Thr Thr Ala Val Pro Asn 115 120 125 Thr Ala Val
Pro Thr Pro Ala Pro Thr Pro Asp Leu Ser Asn Asn Leu 130 135 140 Ala
Ser Arg Cys Ser Thr Gln Leu Gly Met Glu Gly Gly Ala Ile Ala145 150
155 160 Asp Ser Gln Ile Ser Ala Ser Ser Val Tyr Met Gly Phe Met Gly
Leu 165 170 175 Gln Arg Trp Gly Pro Glu Leu Ala Arg Leu Tyr Arg Thr
Gly Ile Val 180 185 190 Asn Ala Trp Thr Ala Ser Asn Tyr Asp Ser Lys
Pro Trp Ile Gln Val 195 200 205 Asn Leu Leu Arg Lys Met Arg Val Ser
Gly Val Met Thr Gln Gly Ala 210 215 220 Ser Arg Ala Gly Arg Ala Glu
Tyr Leu Lys Thr Phe Lys Val Ala Tyr225 230 235 240 Ser Leu Asp Gly
Arg Lys Phe Glu Phe Ile Gln Asp Glu Ser Gly Gly 245 250 255 Asp Lys
Glu Phe Leu Gly Asn Leu Asp Asn Asn Ser Leu Lys Val Asn 260 265 270
Met Phe Asn Pro Thr Leu Glu Ala Gln Tyr Ile Lys Leu Tyr Pro Val 275
280 285 Ser Cys His Arg Gly Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys
Glu 290 295 300 Leu His Gly Cys Ser Glu Pro Leu Gly Leu Lys Asn Asn
Thr Ile Pro305 310 315 320 Asp Ser Gln Met Ser Ala Ser Ser Ser Tyr
Lys Thr Trp Asn Leu Arg 325 330 335 Ala Phe Gly Trp Tyr Pro His Leu
Gly Arg Leu Asp Asn Gln Gly Lys 340 345 350 Ile Asn Ala Trp Thr Ala
Gln Ser Asn Ser Ala Lys Glu Trp Leu Gln 355 360 365 Val Asp Leu Gly
Thr Gln Arg Gln Val Thr Gly Ile Ile Thr Gln Gly 370 375 380 Ala Arg
Asp Phe Gly His Ile Gln Tyr Val Ala Ser Tyr Lys Val Ala385 390 395
400 His Ser Asp Asp Gly Val Gln Trp Thr Val Tyr Glu Glu Gln Gly Ser
405 410 415 Ser Lys Val Phe Gln Gly Asn Leu Asp Asn Asn Ser His Lys
Lys Asn 420 425 430 Ile Phe Glu Lys Pro Phe Met Ala Arg Tyr Val Arg
Val Leu Pro Val 435 440 445 Ser Trp His Asn Arg Ile Thr Leu Arg Leu
Glu Leu Leu Gly Cys 450 455 460 4331PRTArtificial Sequencesynthetic
E1E2D1 mouse milk fat globule epidermal growth factor 8 (Mfge8)
long construct (lacking second discoidin domain) 4Met Gln Val Ser
Arg Val Leu Ala Ala Leu Cys Gly Met Leu Leu Cys1 5 10 15 Ala Ser
Gly Leu Phe Ala Ala Ser Gly Asp Phe Cys Asp Ser Ser Leu 20 25 30
Cys Leu Asn Gly Gly Thr Cys Leu Thr Gly Gln Asp Asn Asp Ile Tyr 35
40 45 Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Val Cys Asn Glu Thr
Glu 50 55 60 Arg Gly Pro Cys Ser Pro Asn Pro Cys Tyr Asn Asp Ala
Lys Cys Leu65 70 75 80 Val Thr Leu Asp Thr Gln Arg Gly Asp Ile Phe
Thr Glu Tyr Ile Cys 85 90 95 Gln Cys Pro Val Gly Tyr Ser Gly Ile
His Cys Glu Thr Glu Thr Asn 100 105 110 Tyr Tyr Asn Leu Asp Gly Glu
Tyr Met Phe Thr Thr Ala Val Pro Asn 115 120 125 Thr Ala Val Pro Thr
Pro Ala Pro Thr Pro Asp Leu Ser Asn Asn Leu 130 135 140 Ala Ser Arg
Cys Ser Thr Gln Leu Gly Met Glu Gly Gly Ala Ile Ala145 150 155 160
Asp Ser Gln Ile Ser Ala Ser Ser Val Tyr Met Gly Phe Met Gly Leu 165
170 175 Gln Arg Trp Gly Pro Glu Leu Ala Arg Leu Tyr Arg Thr Gly Ile
Val 180 185 190 Asn Ala Trp Thr Ala Ser Asn Tyr Asp Ser Lys Pro Trp
Ile Gln Val 195 200 205 Asn Leu Leu Arg Lys Met Arg Val Ser Gly Val
Met Thr Gln Gly Ala 210 215 220 Ser Arg Ala Gly Arg Ala Glu Tyr Leu
Lys Thr Phe Lys Val Ala Tyr225 230 235 240 Ser Leu Asp Gly Arg Lys
Phe Glu Phe Ile Gln Asp Glu Ser Gly Gly 245 250 255 Asp Lys Glu Phe
Leu Gly Asn Leu Asp Asn Asn Ser Leu Lys Val Asn 260 265 270 Met Phe
Asn Pro Thr Leu Glu Ala Gln Tyr Ile Lys Leu Tyr Pro Val 275 280 285
Ser Cys His Arg Gly Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys Lys 290
295 300 Pro Phe Met Ala Arg Tyr Val Arg Val Leu Pro Val Ser Trp His
Asn305 310 315 320 Arg Ile Thr Leu Arg Leu Glu Leu Leu Gly Cys 325
330 5431PRTBos taurusbovine milk fat globule epidermal growth
factor 8 (Mfge8, MFG-E8), lactadherin, sperm surface protein SP47
(BP47), MGP57/53, PAS-6/PAS-7 glycoprotein, componenets 15/16, MFGM
5Met Pro Cys Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Phe Cys1 5
10 15 Ser Ser Gly Leu Phe Ala Phe Ser Gly Asp Phe Cys Asp Ser Ser
Gln 20 25 30 Cys Leu His Gly Gly Thr Cys Leu Leu Asn Glu Asp Arg
Thr Pro Pro 35 40 45 Phe Tyr Cys Leu Cys Pro Glu Gly Phe Thr Gly
Leu Leu Cys Asn Glu 50 55 60 Thr Glu His Gly Pro Cys Phe Pro Asn
Pro Cys His Asn Asp Ala Glu65 70 75 80 Cys Gln Val Thr Asp Asp Ser
His Arg Gly Asp Val Phe Ile Gln Tyr 85 90 95 Ile Cys Lys Cys Pro
Leu Gly Tyr Val Gly Ile His Cys Glu Thr Thr 100 105 110 Cys Thr Ser
Pro Leu Gly Met Gln Thr Gly Ala Ile Ala Asp Ser Gln 115 120 125 Ile
Ser Ala Ser Ser Met His Leu Gly Phe Met Gly Leu Gln Arg Trp 130 135
140 Ala Pro Glu Leu Ala Arg Leu His Gln Thr Gly Ile Val Asn Ala
Trp145 150 155 160 Thr Ser Gly Asn Tyr Asp Lys Asn Pro Trp Ile Gln
Val Asn Leu Met 165 170 175 Arg Lys Met Trp Val Thr Gly Val Val Thr
Gln Gly Ala Ser Arg Ala 180 185 190 Gly Ser Ala Glu Tyr Leu Lys Thr
Phe Lys Val Ala Tyr Ser Thr Asp 195 200 205 Gly Arg Gln Phe Gln Phe
Ile Gln Val Ala Gly Arg Ser Gly Asp Lys 210 215 220 Ile Phe Ile Gly
Asn Val Asn Asn Ser Gly Leu Lys Ile Asn Leu Phe225 230 235 240 Asp
Thr Pro Leu Glu Thr Gln Tyr Val Arg Leu Val Pro Ile Ile Cys 245 250
255 His Arg Gly Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys Glu Leu Asn
260 265 270 Gly Cys Thr Glu Pro Leu Gly Leu Lys Asp Asn Thr Ile Pro
Asn Lys 275 280 285 Gln Ile Thr Ala Ser Ser Tyr Tyr Lys Thr Trp Gly
Leu Ser Ala Phe 290 295 300 Ser Trp Phe Pro Tyr Tyr Ala Arg Leu Asp
Asn Gln Gly Lys Phe Asn305 310 315 320 Ala Trp Thr Ala Gln Thr Asn
Ser Ala Ser Glu Trp Leu Gln Ile Asp 325 330 335 Leu Gly Ser Gln Lys
Arg Val Thr Gly Ile Ile Thr Gln Gly Ala Arg 340 345 350 Asp Phe Gly
His Ile Gln Tyr Val Ala Ala Tyr Arg Val Ala Tyr Gly 355 360 365 Asp
Asp Gly Val Thr Trp Thr Glu Tyr Lys Asp Pro Gly Ser Ser Glu 370 375
380 Ser Lys Ile Phe Pro Gly Asn Met Asp Asn Asn Ser His Lys Lys
Asn385 390 395 400 Ile Phe Glu Thr Pro Phe Gln Ala Arg Phe Val Arg
Ile Gln Pro Val 405 410 415 Ala Trp His Asn Arg Ile Thr Leu Arg Val
Glu Leu Leu Gly Cys 420 425 430 6426PRTCapra hircusgoat milk fat
globule epidermal growth factor 8 (Mfge8, MFG-E8) 6Met Pro Ser Pro
Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Phe Cys1 5 10 15 Ala Ser
Gly Leu Phe Ala Phe Ser Gly Asp Leu Cys Asp Ser Ser Gln 20 25 30
Cys Leu His Gly Gly Thr Cys Leu Leu Asn Glu Glu Lys Thr Pro Pro 35
40 45 Phe Tyr Cys Leu Cys Pro Glu Gly Phe Thr Gly Leu Leu Cys Asn
Glu 50 55 60 Thr Glu Tyr Gly Pro Cys Phe Pro Asn Pro Cys His Asn
Asp Ala Glu65 70 75 80 Cys Gln Val Thr Asp Thr Leu Arg Gly Asp Val
Phe Thr His Tyr Ile 85 90 95 Cys Lys Cys Pro Leu Gly Tyr Val Gly
Thr His Cys Glu Ser Thr Cys 100 105 110 Thr Ser Pro Leu Gly Met Gln
Thr Gly Ala Ile Ala Asp Ser Gln Ile 115 120 125 Ser Ala Ser Ser Met
His Leu Gly Phe Met Gly Leu Gln Arg Trp Ala 130 135 140 Pro Glu Leu
Ala Arg Leu Tyr Gln Thr Gly Ile Val Asn Ala Trp Thr145 150 155 160
Ser Ser Asn Tyr Asp Lys Asn Pro Trp Ile Gln Val Asn Leu Leu Arg 165
170 175 Lys Met Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser Arg Ala
Gly 180 185 190 Ser Ala Glu Tyr Val Lys Thr Phe Lys Val Ala Tyr Ser
Asn Asp Gly 195 200 205 His
Gln Phe Gln Phe Ile Gln Ala Ala Gly Gln Leu Gly Glu Lys Val 210 215
220 Arg Asn Asn Ser Gly Leu Lys Ile Asn Leu Phe Asp Ser Pro Leu
Glu225 230 235 240 Thr Gln Tyr Val Arg Leu Val Pro Ile Ile Cys Arg
Arg Gly Cys Thr 245 250 255 Leu Arg Phe Glu Leu Leu Gly Cys Glu Leu
Asp Gly Cys Thr Glu Pro 260 265 270 Leu Gly Leu Lys Asp Asn Thr Ile
Pro Asn Lys Gln Ile Thr Ala Ser 275 280 285 Ser Tyr Tyr Lys Thr Trp
Gly Leu Ser Ala Phe Ser Trp Phe Pro Tyr 290 295 300 Tyr Ala Arg Leu
Asp Asn Trp Gly Lys Phe Asn Ala Trp Thr Ala Gln305 310 315 320 Thr
Asn Ser Ala Ser Glu Trp Leu Gln Ile Asp Leu Gly Ser Gln Lys 325 330
335 Arg Val Thr Gly Ile Ile Thr Gln Gly Ala Arg Asp Phe Gly His Ile
340 345 350 Gln Tyr Val Ala Ala Tyr Arg Val Ala Tyr Ser Asp Asp Gly
Val Thr 355 360 365 Trp Thr Glu Tyr Lys Asp Pro Glu Thr Ser Lys Ser
Lys Ile Phe Pro 370 375 380 Gly Asn Met Asp Asn Asn Ser His Lys Lys
Asn Ile Phe Glu Val Pro385 390 395 400 Phe Gln Ala Arg Phe Val Arg
Ile Gln Pro Val Ala Trp His Asn Arg 405 410 415 Ile Thr Leu Arg Val
Glu Leu Leu Gly Cys 420 425 76PRTArtificial Sequencesynthetic
polyhistidine affinity purification fusion domain, detection
domain, His6 7His His His His His His1 5
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