BETA-LIPOTROPIN AND USES THEREOF
CROSS-REFERENCE
This application claims the benefit of U.S. Provisional
Application Nos. 60/068,659, filed December 23, 1997;
60/079,857, filed March 30, 1998; 60/086321, filed May 21,
1998; 60/091,385, filed July 1, 1998; 60/095,405, filed
August 5, 1998; and 60/103,976, filed October 13, 1998.
BACKGROUND OF THE INVENTION
This invention relates to the pharmaceutical and
medical arts. In particular the invention pertains to beta-
lipotropin, fragments and analogs thereof, pharmaceutical
formulations, and methods for using same in treating
diabetes and other associated conditions in mammals.
Proopiomelanocortin (POMC) is a neuropeptide precursor
molecule which is translocated to secretory pathways withdn
neuroendocrine cells. POMC is cleaved by the action of
specific endopeptidases to yield peptides such as
adrenocorticotrophic hormone (ACTH) , Beta-lipotropic (BLT"> ,
Beta-endorphin, and Melanocyte Stimulating Hormone (MSH).
The orocessing of POMC into one or more specific peptides
occurs in a tissue and cell specific manner (See generally,
:•'.. Castro and E. Morrison, Crit. Rev. Neurobiol. , 11, 35-57,
1957; Roberts, J. L. and Herbert, E., Proc Naz Acad Sci, 7-1,
4825 (1977): Roberts, J. L. and Herbert, E.,
Proc.Nat.Acad.Sci 74, 5300 (1977); Mains, et al.,
Proc.Nat.Acad.Sci 74, 3014 (1977)). POMC is produced mainly
in the pituitary gland and hypothalamus. Post-translational
processing of POMC in the anterior pituitary produces ACTH
and BLT. On the other hand, the major products of the
intermediate pituitary are cx-MSH, CLIP, y-lipotropin, p-
endorphin, and P-MSH, while in the hypothalamus, POMC is
processed primarily into y-MSH and p-endorphin.
POMC-derived peptides perform a variety of important
roles in metabolic and physiological regulation. For
example, ACTH, a 39 amino acid peptide, stimulates secretion
of glucocorticoids from the adrenal cortex. The MSH's, on
the other hand, stimulate melanin synthesis by melanocytes
in the skin, and also appear to be involved in fat
metabolism, p-endorphin derives from the carboxyl end of BLT
{viz. Residues 59 to 89 of the human sequence), and
possesses analgesic activity that is antagonized by
naloxone, a known antagonist for morphine. Thus, POMC-
derived peptide hormones have diverse roles in physiologic
and metabolic regulation.
Proper glucose and fuel metabolism depend on the non-
POMC related peptide, insulin. Specifically, insulin
stimulates glycogen, fatty acid, and protein synthesis, and
also stimulates glycolysis. Insulin is critical in promoting
entry of glucose into muscle and fat cells.
Defective insulin metabolism may lead to diabetes. Type
i diabetics require exogenous insulin administration for
proper control of fuel and glucose metabolism. On the other
hand, Type 2 diabetics typically do not require exogenous
insulin until the later stages of the disease. Proper
control of glucose and fuel metabolism is essential for
effective management of diabetes. Without this, there can be
serious, perhaps even fatal, consequences including
ketoacidosis, coma, retinopathy, diabetic microangiopathy,
atherosclerosis, myocardial infarction, stroke, gangrene,
hypertriglyceridemia, hypercholesterolemia, cardiomyopathy,
dermopathy, diabetic foot syndrome, nephropathy, unrinary
tract infection, papillary necrosis, cataracts, diabetic
gastroenteropathy, constipation, peripheral vascular
disease, and even death. In many instances, a delicate
balance must be struck between administration of too much
insulin and too little insulin. Therefore, an ideal therapy
for diabetes would be one that controls blood glucose levels
by improving sensitivity to insulin.
Described herein is a method for treatment and
pharmaceutical composition that is effective in treating or
preventing type 1 and type 2 diabetes, and associated
complications thereof, comprising the administration of a
pharmaceutically effective amount of beta-lipotropin and/or
fragments and/or analogs thereof.
BRIEF SUMMARY OF THE INVENTION
The present invention, provides isolated proteins
comprising beta-lipotropin (3LT), analogs thereof, fragments
thereof, nucleic acids encoding same, and methods for
producing and using BLT in the treatment of diabetes and
complications associated therewith.
Disclosed herein are methods for treating diabetes and
complications thereof in mammals, including humans, by
administering pharmaceutically effective amounts of BLT,
analogs thereof, or functional fragments thereof. The
invention relates further to methods of treatment for type 1
and type 2 diabetes, retinopathy, diabetic microangiopathy,
atherosclerosis, myocardial infarction, stroke, gangrene,
hypertriglyceridemia, hypercholesterolemia, cardiomyopathy,
dermopathy, diabetic foot syndrome, nephropathy, unrinary
tract infection, papillary necrosis, cataracts, diabetic
gastroenteropathy, constipation, and peripheral vascular
disease.
Disclosed herein are methods for producing BLT in E.
coli and yeast. In E. coli BLT is produced as a fusion
protein which can be recovered from cell lysates in the
presence of high salt by conventional purification methods.
The BLT fusion protein contains a recognition site for
specific proteases which are used to separate the fusion
partner from BLT. In the yeast Pichia pastoris, the fusion
protein is cleaved upon secretion of the protein from the
cell such that native BLT can be recovered intact from the
culture medium.
In one embodiment the present invention relates to a
substantially pure protein having the amino acid sequence
which is SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 7.
In still another embodiment the present invention rslaces sc-
an isolated nucleic acid compound encoding a fusion protein cr
recombinant protein or peptide comprising beta lipotropic.
In another embodiment the present invention relates tc at
least one isolated nucleic acid compound encoding a protein cr
peptide of the present invention.
In still another embodiment the present invention
relates to a vector comprising an isolated nucleic acid
compound of the invention.
In yet another embodiment the present; invention relates
to a vector comprising an isolated nucleic acid of the
invention, wherein said isolated nucleic acid compound is
operably-linked to a promoter sequence.
In another embodiment the present invention relates to
a host cell containing a vector of the present invention.
In yet another embodiment the present invention relates
to a method for constructing a recombinant host cell having
the potential to express beta-lipotropin, said method
comprising introducing into said host cell by any suitable
means a vector of the invention.
In still another embodiment the present invention
relates to a method for expressing beta-lipotropin in a
recombinant host cell, said method comprising culturing said
recombinant host cell under conditions suitable for gene
expression.
In another embodiment the present invention relates to
a pharmaceutical formulation comprising as an active
ingredient beta-lipotropin, analog, or functional fragment
thereof, associated with one or more pharmaceutically
acceptable carriers, excipients, or diluents therefor.
In still another embodiment the present invention
relates to a pharmaceutical formulation wherein said be~a-
lipotropin, analog, or functional fragment thereof, is human
beta-lipotropin.
In yet another embodiment the present invention relates
to beta-lipotropin, analog, or functional fragment thereof,
for use in treating diabetes or complications thereof.
In still another embodiment the present invention
relates to fragments of BLT having insulinotropic activity.
In still another embodiment the present invention
relates to a peptide having insulinotropic activity selected
from the group consisting of SEQ ID NO:9 through SEQ ID
NO:13.
In still another embodiment the present invention
relates to a peptide having insulinotropic activity selected
from the group consisting of SEQ ID NO:14 through SEQ ID
NO:25.
In still another embodiment the present invention
relates to a functional analog of a beta-lipotropin peptide
disclosed herein.
In still another embodiment the present invention
relates to a method for treating diabetes comprising
administration of a therapeutically effective amount of at
least one peptide selected from the group consisting of SEQ
ID NO:9 through SEQ ID NO:25.
In still another embodiment the present invention
relates to a method for treating diabetes comprising
administration of a therapeutically effective amount of a
peptide selected from the group consisting of SEQ ID NO:9
through SEQ ID NO:13.
In still another embodiment the present invention
relates to a pharmaceutical formulation comprising as an
active ingredient at least one peptide having insulinotropic
activity selected from the group consisting of SEQ ID NO:3
through SEQ ID NO:25.
In still another embodiment the present invention
relates to a method for treating diabetes in a mammal
including a human comprising administration of a
therapeutically effective amount of beta-lipotropin, analog,
or functional fragment thereof.
In yet another embodiment the present invention relates
to a method for lowering blood glucose levels in a manmal by
administering an effective amount of beta-lipotropin,
analog, or functional fragment thereof.
In yet another embodiment the present invention relates
to a method for treating hyperglycemia in a mammal in need
thereof by administering an effective amount of beta-
lipotropin, analog, or functional fragment thereof.
In still another embodiment the present invention
relates to a method for treating hyperinsulinemia in a
mammal by administering an effective amount of beta-
lipotropin, analog, or functional fragment thereof.
In another embodiment the present invention relates to
a method for enhancing insulin sensitivity in a mammal by
administering an effective amount of beta-lipotropin,
analog, or functional fragment thereof.
In another embodiment the present invention relates to
a solid-phase synthetic method for synthesizing beta-
lipotropin, analog, or functional fragment thereof.
In another embodiment the present invention relates to
a process for preparing beta-lipotropin comprising:
a. transforming a suitable host with an expression
vector wherein said vector encodes a beta-lipotropin,
analog, or functional fragment thereof;
b. culturing said transformed host under conditions
that enable expression of said beta-lipotropic;
c. purifying said beta-lipotropin by any suitable
means.
In yet another embodiment the present invention relates
to an assay for beta-lipotropic activity comprising the
steps of:
a) administering tc a mammal that exhibits insulin
insensitivity and elevated blood glucose levels a test
protein; and
b) testing for blood glucose and insulin levels
after step (a).
The invention also relates to a method of treating type
1 or type II diabetes and complications associated therewith
in mammals by the administration of a pharraaceutically
effective amount of beta-lipotropin, analog, or functional
fragment thereof.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term "analog" or "functional analog" refers to a
modified form of BLT in which at least one amino acid
substitution has been made such that said analog retains
substantially the same biological activity as the unmodified
BLT in vivo and/or in vitro.
The term "bid" or "b.i.d." refers to a dosage of ELT or
other compound administered twice daily.
"3LT" refers to beta-lipotropin. The human BLT
comprises 89 amino acid residues (SEQ ID NO:8). The BLT
protein has been characterized in a variety of organisms and
the ar.ino acid sequences determined in a variety of
organisms including human, mouse, ovine, and porcine (Li &
Chung, Nature, 260, 622-24 (1976); and elephant (Li et al.
Int. J. Pept. Prot. Res. 32, 573-78, 1S88); as well as other
mammals, all of which are hereby incorporated by reference.
The term "beta-lipcprctein fusion protein" or "BLT
fusion protein" refers tc a class of hybrid recombinant:
protein molecules comprising BLT, that are produced in E.
coli or other cell type and from which can be generated BLT,
or a BLT fragment through specific proteolysis or chemical
cleavage. Exemplary BLT fusion proteins include those
specified herein as SEQ ID NO-.2, SEQ ID NO: 5, and SEQ ID
NO: 7.
The terms "complementary" or "complementarity'' as
used herein refer to the capacity of purine and pyrir.idine
nucleotides to associate through hydrogen bonding to form
double stranded nucleic acid molecules. The following base
pairs are related by complementarity: guanine and cytosine;
adenine and thymine; and adenine and uracil. As used herein,
"complementary" means that the aforementioned relationship
applies to substantially all base pairs comprising two
single-stranded nucleic acid molecules over the entire
length of said molecules. "Partially complementary" refers
to the aforementioned relationship in which one of two
single-stranded nucleic acid molecules is shorter in length
than the other such that a portion of one of the molecules
remains single-stranded.
The term "complications" or "complications
thereof" as used herein refers to conditions, syndromes.,
ancillary diseaese(s), ailments, or the like associated with
one or more diseases or syndromes, or conditions associated
with defective insulin metabolism, or defective carbohydrate
metabolism, e.g. defective glucose metabolism, including but
not limited to type 1 and type 2 diabetes. Examples of
complications would include ketoacidosis, coma, retinopathy,
diabetc microangiopathy, atherosclerosis, myocardial
infarction, stroke, gangrene, hypertriglyceridemia,
hypercholesterolemia, cardiomyopathy, dermopathy, diabetic
foot syndrome, nephropathy, unrinary tract infection,
papillary necrosis, cataracts, diabetic gastroenteropathy,
constipation, and peripheral vascular disease.
"Conservative substitution" or "conservative amino
acid substitution" refers to a replacement of one or mere
arr.ino acid residue (s) in a protein or peptide as exemplified
in Table 1.
"Fragment thereof" refers to a fragment, piece, or
sub-region of a peptide, or a nucleic acid, such that the
fragment comprises 2 (two) or more contiguous amino acids,
or alternatively about 5 to 14 amino acids, or greater; or
10 or more nucleotides that are contiguous in the parent
peptide or nucleic acid molecule. Fragment thereof may or
may not retain biological activity. For example, a fragnent
of a peptide disclosed herein could be used as an antigen to
raise a specific antibody against the parent peptide frcm
which the fragment is derived, when referring to a nucleic
acid molecule, "fragment thereof" refers to 10 or more
contiguous nucleotides, derived from the parent nucleic
acid, and also, owing to the genetic code, to the
complementary sequence. For example if the fragment entails
the sequence 5'-AGCTAG-3', then "fragment thereof" would
also include the complementary sequence, 3'-TCGATC-5'.
The term "fusion protein" denotes a hybrid protein
molecule not found in nature comprising a translational
fusion or enzymatic fusion in which two or more different
proteins or fragments thereof are covalently linked on a
single polypeptide chain.
"Fusion partner" refers to a sequence of amino
acids in a BLT fusion protein wherein said sequence does not
derive from BLT, e.g. the sequence identified herein as SEQ
ID NO:6.
"Functional fragment" or "functionally equivalent
fragment", as used herein, refers to a region, or fragment
of a full length BLT that retains biological activity, i.e.
the ability to enhance the effect of insulin in vivo and/or
in vitro; and/or the ability to promote a lowering of blood
glucose in vivo, or to enhance glucose uptake into a cell or
tissue in vitro. Functional fragments may also provide the
biological activity manifested by a full length BLT, in vivo
and/or in vitro, viz. the capacity to promote glucose uptake
and/or to enhance the effects of insulin and/or to enhance
insulin sensitivity. Functional fragments may be produced by
cloning technology, or by chemical synthetic means or by
chemical or enzymatic cleavage.
"Host cell" refers to any eucaryotic or
procaryotic cell that is suitable for propagating and/or
expressing a cloned gene contained on a vector that is
introduced into said host cell by, for example,
transformation or transfection, or the like.
The term "homolog" or "homologous" describes the
relationship between different nucleic acid molecules or
amine acid sequences in which said sequences or molecules
are related by identity or partial identity and/or
similarity at one or more blocks or regions of sequence
within said molecules.
The term "hybridization" as used herein refers to
a process in which a single-stranded nucleic acid molecule
joins with a complementary strand through nucleotide base
pairing. "Selective hybridization" refers to hybridization
under conditions of high stringency. The degree of
hybridization depends upon, for example, the degree of
homology, the stringency of hybridization, and the length of
hybridizing strands.
The term "insulinotropic" refers to an insulin
enhancing activity, for example, by reversing or relieving
the effects of insulin insensitivity.
"Isolated nucleic acid compound" refers to any RNA
or DNA sequence, however constructed or synthesized, which
is locationally distinct from its natural location, e.g. in
a cell.
A "nucleic acid probe" or "probe" as used herein
is a labeled nucleic acid compound which hybridizes with
another nucleic acid compound. "Nucleic acid probe" means a
single stranded nucleic acid sequence that will combine with
a complementary or partially complementary single stranded
target nucleic acid sequence to form a double-stranded
molecule. A nucleic acid probe may be an oligonucleotide or
a nucleotide polymer. A probe will usually contain a
detectable moiety which may be attached to the end(s) of the
probe or be internal to the sequence of the probe.
The term "plasmid" refers to an extrachromosomal
genetic element. The plasmids disclosed herein are
commercially available, publicly available on an
unrestricted basis, or can be constructed from readily
available plasmids in accordance with published procedures.
A "primer" is a nucleic acid fragment which
functions as an initiating substrate for enzymatic or
synthetic elongation of, for example, a nucleic acid
molecule.
The term "promoter" refers to a nucleic acid
sequence that directs transcription, for example, of DNA to
RNA. An inducible promoter is one that is regulatable by
environmental signals, such as carbon source, heat, or metal
ions, for example. A constitutive promoter generally
operates at a constant level and is not regulatable.
The terms "protein" and "peptide" are used
interchangeably throughout, refering to two or more amino
acid residues covalently linked by peptide bonding. In some
instances these terms describe amino acid biopolymers
comprising greater than 10 and up to about 500 amino acid
residues linked by peptide bonding.
"Recombinant DNA cloning vector" as used herein
refers to any autonomously replicating agent, including, but
not limited to, plasmids and phages, comprising a DNA
molecule to which one or more additional DNA segments can or
have been incorporated.
The term "recombinant DNA expression vector" or
"expression vector" as used herein refers to any recombinant
DNA cloning vector, for example a plasmid or phage, in which
a promoter and other regulatory elements are present thereby
enabling transcription of an inserted DNA, which may encode
a protein.
The term "stringency" refers to hybridization
conditions. High stringency conditions disfavor non-
homologous basepairing. Low stringency conditions have the
opposite effect. Stringency may be altered, for example, by-
temperature and salt concentration.
"Low stringency" conditions comprise, for example,
a temperature of about 3 7° C or less, a formamide
concentration of less than about 50%, and a moderate to low
salt (SSC) concentration; or, alternatively, a temperature
of about 50° C or less, and a moderate to high salt (SSPS)
concentration, for example 1M NaCl.
"High stringency" conditions comprise, for
example, a temperature of about 42° C or less, a formamide
concentration of less than about 20%, and a low salt (SSC)
concentration; or, alternatively, a temperature of about 65°
C, or less, and a low salt (SSPE) concentration. For
example, high stringency conditions comprise hybridization
in 0.5 M NaHP04, 7% sodium dodecyl sulfate (SDS) , 1 mM EDTA
at 65°C (Ausubel, F.M. et al. Current Protocols in
Molecular Biology, Vol. I, 1989; Green Inc. New York, at
2.10.3).
"SSC" comprises a hybridization and wash solution.
A stock 2OX SSC solution contains 3M sodium chloride, 0.3M
sodium citrate, pH 7.0.
"SSPE" comprises a hybridization and wash
solution. A IX SSPE solution contains 180 mM NaCl, 9mM
Na;H?0., 0.9 mM NaH2P04 and 1 mM EDTA, pH 7.4.
"Substantially pure" in reference to a protein
means that said protein is separated from other cellular arid
non-cellular components, including other protein molecules.
A substantially pure preparation is at least 85% pure; and
preferably about at least 95% pure. A "substantially pure"
protein could be prepared by any number of suitable methods
including, for example, the IMAC protein purification method
(U.S. Patent No. 4,569,794) herein incorporated by
reference.
"Treating" as used herein describes the management
and care of a patient for the purpose of combating a
disease, condition, or disorder and includes the
administration of a protein of the present invention to
prevent the onset of the symptoms or complications,
alleviating the symptoms or complications, or eliminating
the disease, condition, or disorder.
All references cited in this specification are
herein incorporated by reference.
Beta-lipotropin was isolated in 1964 from ovine
pituitary glands, and its primary structure reported the
following year (Li et. al. Nature, 208, 1093, 1965). Since
then the primary sequence of sheep, bovine, ovine, mouse,
porcine, guinea pig, rat, elephant, and human BLTs have been
reported. (See e.g. Lohmar and Li, Biochim. Biophys. Acta,
147, 381, 1967; Li and Chung, .Nature, 260, 622, 1976; Drouin
and Goodman, Nature, 288, 619, 1980; Li et al. Int. J. Pept.
Prot. Res. 32, 573-78, 1988; Blake and Li, Proc. Nat. Acad.
Sci. 80, 1556-1559, 1983; Takahashi et.al. FEBS Lett. 135,
97-102, 1981; all of which are herein incorporated by
reference). The sequence daca reveal that the carboxy
terminus of BLT is highly conserved across species. On the
ether hand, considerable sequence differences occur at the
amino terminal end of BLT molecules from different species.
The present invention relates further to BLT fusion
proteins, comprising human BLT (SEQ ID NO:8), or BLT from
another species, or a functional fragment thereof.
Exemplary BLT fusion proteins are disclosed herein as SEQ IB
NO: 2, SEQ ID NO:5, and SEQ ID NO:7.
Functional fragments of BLT are conveniently identified
as fragments of BLT that exhibit biological activity, for
example, the capacity to ameliorate diabetic symptoms when
administered to a mammal in need thereof, or to diminish
serum insulin levels, and/or enhance insulin sensitivity,
and/or lower blood glucose levels, and/or stimulate glucose
uptake in adipose or muscle tissue, in vivo or in vitro., or
in adipocytes in vitro. Functional fragments of BLT comprise
any fragment that retains biological activity and that
comprises at least two (2) or more amino acid residues that
are in some instances contiguous in the BLT protein.
Preferred fragments comprise a contiguous region of BLT
mapping partly or wholly outside the region of residues 59
through 89 of human BLT (SEQ ID NO:8), or equivalent region
of non-human BLT (i.e. region encoding B-endorphin)
Exemplary functional fragments of human BLT are
disclosed herein as SEQ ID NO:9 through SEQ ID NO:25.
Preferred fragments are designated herein as SEQ ID NO:9
through SEQ ID NO:13; most preferred fragments are
designated herein as SEQ ID NO:10, SEQ ID NO:12, and SEQ ID
NO:13. In some instances, a functional fragment may comprise
an internal deletion of the parent BLT, e.g. SEQ ID NO:13,
in which amino acid residues 7 through 2 3 of human BLT are
deleted. Functional fragments may be produced by solid phase
chemical synthesis and/or by recombinant DNA techniques well
known to the skilled artisan. See e.g. K. Struhl, "Reverse
biochemistry: Methods and applications for synthesizing
yeast proteins in vitro," Meth. Enzymol. 194, 520-535. For
example, in one method, a nested set of deletion mutations
are introduced into a nucleic acid encoding BLT such that
varying amounts of the peptide coding region are deleted,
either from the amino terminal end, or from the carboxyl end
of the molecule. This method can also be used to create
internal fragments of the intact protein in which both the
carboxyl and amino terminal ends are removed. Suitable
nucleases for creating such deletions include, for example
5a231, or in the case of a single stranded nucleic acid
molecule, mung bean nuclease. For simplicity, it is
preferred that the BLT encoding nucleic acid be cloned into
a single-stranded cloning vector, such as bacteriophage M13,
or equivalent. If desired, the resulting deletion fragments
can be subcloned into any suitable vector for propagation
and expression in any suitable host cell.
Functional fragments of BLT may be identified and
tested for biological activity using any suitable assay, for
example, the ability of a peptide fragment to stimulate or
enhance insulin sensitivity and/or glucose uptake by cells
in vivo or in vitro.
Functional analogs of BLT can be generated by deletion,
insertion, inversion, and/or substitution of one or more
amino acid residues in said BLT, cr any one cf the peptides
disclosed herein. Substitution analogs can generally be made
by solid phase or recombinant techniques in which single or
multiple conservative amino acid substitutions are made, for
example, according to Table 1. Generally, in the case of
multiple substitutions, it is prefered that less than ten
residues be changed in any given molecule, most preferably
between one to five residues are changed in any given
molecule such that about between 90% to 99% of residues are
identical with the sequence of SEQ ID NO:8; alternatively,
such that about between 95% to 99% of residues are identical
with SEQ ID NO:8 or other suitable BLT from another species.
For example, analogs of human BLT (SEQ ID NO:8)
comprising single or multiple amino acid substitutions in
the region between amino acid residues l through 89,
comprise substitutions wherein:
the residue at position 1 is alternatively Glu, Ala,
Asp or Gin;
the residue at position 2 is alternatively Leu, lie.
Val, or Met;
the residue at position 3 is alternatively Thr, Ala,
Glu, Ser, Pro, or Gly;
the residue at position 4 is alternatively Gly, Arg,
Ala, Leu, Pro, or Ser;
the residue at position 5 is alternatively Glu, Gin,
Asp, Asn, or Ala;
the residue at position 6 is alternatively Arg, Glu,
Leu, Lys, Gin, or Ala;
the residue at position 7 is alternatively Leu, Pro,
Asp, Val, lie, or Met;
the residue at position 8 is alternatively Arg, Glu,
Ala, Tyr, Leu, Lys, Pro, Gin or Trp;
the residue at position 9 is alternatively Glu, Ala,
Pro, Asp, Asn, or Gin;
the residue at position 10 is alternatively Gly, Ala,
Ser, or Asp;
the residue at position 11 is alternatively Asp, Arg,
Pro, Asn, Gin, Ala, or Glu;
the residue at position 12 is alternatively Gly, Ala,
Ser, or Met;
the residue at position 13 is alternatively Pro, Glu,
Gly, or Val;
the residue at position 14 is alternatively Asp, Glu,
Asn, Gin, or Gly;
the residue at position 15 is alternatively Gly, Ala,
Ser, or Glu;
the residue at position 16 is alternatively Pro, Gin,
Leu, Gly, or Glu;
the residue at position 17 is alternatively Ala, Asp,
Ser, or Gly;
the residue at position 18 is alternatively Asp, Glu,
Gin, or Asn;
the residue at position 19 is alternatively Asp, Glu,
Asn, or Gin;
the residue at position 20 is alternatively Gly, Ser,
or Ala;
the residue at position 21 is alternatively Ala, Gly,
Ser, or Phe;
the residue at position 22 is alternatively Gly, Ala,
Ser, or Lys;
the residue at position 23 is alternatively Ala, Phe,
Thr, Gly, Ser, or Leu;
rhe residue at position 24 is alternatively Gin, Arg,
Asp, Asn, Leu, or Val;
the residue at position 25 is alternatively Ala, Leu,
Asp, lie, Gly, Ser, or Thr;
the residue at position 26 is alternatively Asp, Glu,
Gly, Asn, Gin, or Lys;
the residue at position 27 is alternatively Leu, Ala,
lie. Met, or Val;
the residue at position 2 8 is alternatively Glu, Gin,
Asn, or Asp;
the residue at position 2 9 is alternatively His, Asn,
Tyr, Ala, Gin or Glu;
the residue at position 3 0 is alternatively Ser, Gly,
Glu, Ala, Leu, or Asp;
the residue at position 31 is alternatively Leu, Ala,
Val, Met, or lie;
the residue at position 32 is alternatively Leu, Ala,
Val, lie, Met, or Pro;
the residue at position 33 is alternatively Val, Ala,
Glu, Leu, lie. Met, or Arg;
the residue at position 34 is alternatively Ala, Ser,
Pro, Glu, or Gly;
the residue at position 35 is alternatively Ala, Asp,
Gly, Ser, or Leu;
the residue at position 36 is alternatively Glu, Ala,
Thr, Leu, Asp, Asn, or Gin;
the residue at position 37 is alternatively Lys, Glu,
Thr, Arg, Gin, or Asp;
the residue at position 3 8 is alternatively Lys, Arg,
Gin, or Glu;
the residue at position 3 9 is alternatively Asp, Ala,
Asn, Glu, Gin, or Lys,-
the residue at position 4C is alternatively Glu, Ser,
Asp, Asn, Gin, or Gly;
the residue at position 41 is alternatively Gly, Ala,
or Ser;
the residue at position 42 is alternatively Pro, Gly,
Ser, or Asn;
the residue at position 43 is alternatively Tyr, Phe,
or Trp;
the residue at position 44 is alternatively Arg, Lys,
Gin, or Glu;
the residue at position 45 is alternatively Met, lie,
Ser, or- Val;
the residue at position 46 is alternatively Glu, Gin,
Asp, Asn, His, Arg or Gly;
the residue at position 4 8 is alternatively Tyr or ?rp;
the residue at position 49 is alternatively Arg cr Lys;
the residue at position 50 is alternatively Trp, Tyr,
or Phe;
the residue at position 51 is alternatively Gly, Ala,
Ser, or gin;
the residue at position 52 is alternatively Ser, Thr,
Asn, or Ala;
the residue at position 53 is alternatively Pro or Gly;
the residue at position 54 is alternatively Pro, Ala,
Gly, Arg, Leu, or Thr;
the residue at position 55 is alternatively Lys, Arg,
Gin, or Ala;
the residue at position 56 is alternatively Asp, Asn,
Glu, Gin, Ala, Gly, or lie;
the residue at position 87 is alternatively Lys, Gin,
or Arg;
the residue at position 88 is alternatively Gly, Ala,
or Ser;
the residue at position 89 is alternatively Glu, Gin,
Asp, Asn, or His.
Additional specific substitutions in human BLT (SEQ ID
NO:8) include single or multiple substitutions in SEQ ID
NO:8, wherein:
the residue at position 3 is Glu;
the residue at position 4 is Arg;
the residue at position 6 is Gin;
the residue at position 7 is Pro;
the residue at position 8 is Glu, Ala, or Pro;
the residue at position 9 is Pro or Ala;
the residue at position 11 is Arg or Pro;
the residue at position 16 is Leu or Gin;
the residue at position 21 is Phe;
the residue at position 23 is Thr, Leu or Pro;
the residue at position 24 is Arg or Val ,-
the residue at position 2 7 is Ala;
the residue at position 2 9 is Asn, Tyr, or Ala;
the residue at position 3 0 is Glu;
the residue at position 31 is Ala;
the residue at position 32 is Ala;
the residue at position 3 3 is Ala or Glu;
the residue at position 34 is Pro or Ser;
the residue at position 35 is Asp;
the residue at position 3 6 is Thr or Ala;
the residue at nosition 37 is Glu;
Gene Isolation Procedures
Those skilled in the art will recognize that a nucleic
acid encoding BLT, or a BLT fusion, can be obtained by a
plurality of recombinant DNA techniques including, for
example, polymerase chain reaction (PCR) amplification, or
de novo DNA synthesis.(See e.g., T. Maniatis et al.
Molecular Cloning: A Laboratory Manual, 2d Ed. Chap. 14
(1989)).
For example, oligonucleotide primers targeted to the 3'
and 5' ends of SEQ ID NO:l can be used for PCR amplification
of Met-Arg-BLT. See e.g. PCR Protocols: A Guide to Method
and Application, Ed. M. Innis et al., Academic Press (1990}.
A PCR amplification comprises template DNA, suitable
enzymes, primers, and buffers, and is conveniently carried
out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk,
CT). A positive result is determined by detecting an
appropriately-sized DNA fragment (viz. 273 base pairs)
following agarose gel electrophoresis.
Protein Production Methods
One embodiment of the present invention relcites :o
the use of BLT protein as a pharmaceutical compound.
Skilled artisans will recognize that the proteins
and fragments, or functional fragments thereof, of the
present invention can be synthesized by a number of
different methods, such as chemical methods well known in
the art, including solid phase peptide synthesis or
recombinant methods. Both methods are described in U.S.
Patent 4,617,14 9, incorporated herein by reference.
The principles of solid phase chemical synthesis
of proteins are well known in the art and may be found in
general texts in the area. See, e.g., H. Dugas and C.
Penney, Bioorganic Chemistry (1981) Springer-Verlag, New
York, 54-92. For example, peptides may be synthesized by
solid-phase methodology utilizing an Applied Biosystems 430A
peptide synthesizer (Applied Biosystems, Foster City, CA)
and synthesis cycles supplied by Applied Biosystems.
Sequential t-butoxycarbonyl chemistry using
double-couple protocols are applied to the starting p-methyl
benzhydryl amine resins for the production of C-terminal
carboxamides. For the production of C-terminal acids, the
corresponding pyridine-2-aldoxime methiodide resin is used.
Asparagine, glutamine, and arginine are coupled using
preformed hydroxy benzotriazole esters. Following completion
of the synthesis the peptides may be deprotected and cleaved
from the resin with anhydrous hydrogen fluoride containing
10% meta-cresol. Cleavage of the side chain protecting
group (s) and of the peptide from the resin is carried out at
zero degrees Celsius or below, preferably -20°C for thirty
minutes followed by thirty minutes at 0°C.
In general, the synthesis of a peptide consists of a
series of steps as follows. First, an amino acid with its a-
amino (and side chain functional group if necessary)
protected is activated to form a reactive ester. This
activated amino acid is attached to an inert solid support
through this activated ester croup and the bound amino acid
is thoroughly washed. Following this step, a second
protected amino acid is activc-ted to an ester in a separate
reaction. The a-amino group of the first amino acid :-:; de-
protected yielding a reactive amine, and the second
activated amino acid is reacted with it to form a di-peptide
on the solid support. Sequential repetition of these steps
results in a peptide of increasing length. Following
completion of the synthesis, the peptide is removed from the
solid support and the side chain functional groups are de-
protected by treatment of the peptide with a strong acid.
The peptide is then separated from the solid support by
filtration, precipitated with an organic solvent, and
¦purified by a variety of techniques well known in the art.
In the present instance, the a-amino protecting group
is the 9-fluoroenylmethylcarbcnyl (Fmoc) group. Side chain
protecting groups are; Boc (for Lys and Trp residues) , Trt
(for Asn, His, Gin) , tBu (for Ser, Thr, Tyr) , OtBu (for Asp,
Glu 'i and Pmc (for Arg) . Active esters are formed with 2-
(lH-benzo-triazole-1-yl)-1,1,3,3-tetramethyl uronium
hexafluorophosphate (HBTU).
The a-amino Fmoc protecting group is removed by
treatment of the solid phase with piperidine. Under these
conditions the Fmoc group is readily removed while the solid
phase linkage and side chain protecting groups are no;
effected. The protected amino acid to be added to a nascent
peptide chain is activated with HBTU.
The synthesis of human BIjT presented several special
challenges. First, the sequence of the peptide contains a
ser-pro-pro tripeptide segment. Standard coupling chemistry
results in a series of deletion errors in the synthesis.
Such deletions are minimized by double-coupling the pro
residues. In the preferred embodiment the synthesis is;
carried out by double-coupling the Pro and Ser residues. In
addition, after completion of the coupling step, any
remaining unreacted, deprotected peptide is blocked with
acetic anhydride to prevent chain lengthening of a deletion
peptide.
Second, the human peptide contains 3 asp-gly dipeptide
sequences. These sequences are susceptible to cyclic imine
formation, even when the asp side chains are protected.
These cyclic imines may tnen react with piperidine in the
deprotections step to generate a piperidine-modified
peptide. This cyclization was eliminated by utilization of
N-a-Hmb protection of each glycine residue preceding an asp
residue. See e.g. Qubbell et.al. J. Chem. Soc. Chem. Commun.
2343, 1994, herein incorporated by reference. Two of the
asp-gly dipeptides are preceded by a pro. Due to this
feature of the sequence, and the lower reactivity of the hmb
protected amino acids, each of these are multiply-coupled,
and then capped with acetic anhydride. In a preferred
embodiment these are double-coupled.
In a preferred method the peptide is synthesized
in a single run using Fmoc chemistry. The synthesis of BLT
is complicated by the presence of several asp-gly dipeptide
sequences in the N-terminal portion of the molecule.
Aspartyl side chains in asp-gly didpeptide sequences have
been observed to undergo base catalyzed cyclization and
subsequent addition with piperidine during FMOC synthesis.
This reaction is eliminated by use of Fmoc- (FmocHmb) -glycine
at each asp-gly sequence in the synthesis. Protection of the
glycyi amide with the Hmb group inhibits the cyclization of
the aspartyl side chain. Following cleavage, deprocection,
and reverse phase HPLC purification, the peptide can be
analyzed by electospray mass spectometry. The major species
seen in this synthesis of 3LT is the full length peptide
having the expected mass. Use of this method allows
production of quantities of purified protein in excess of
100 mg from a single run at the 0.1 mmole scale.
The proteins of the present invention can also be
produced by recombinant DNA methods. Expression of a cloned
nucleic acid encoding BLT, or BLT fusion (e.g. SEQ ID NO:l),
can be carried out in a variety of suitable host cells, well
known to those skilled in the art. For this purpose, a BLT
encoding nucleic acid is introduced into a host cell by any
suitable means, well known to those skilled in the art.
While chromosomal integration is within the scope of the
present invention, it is preferred that the sequence be
cloned into a suitable extra-chromosomally maintained
expression vector so that the coding region of the BLT gene
is operably-linked to a constitutive or inducible promoter.
The basic steps in the recombinant production of
the BLT protein are:
a) constructing a natural, synthetic or
semi-synthetic DNA encoding BLT, or a fusion
protein thereof,-
b) integrating said DNA into an expression
vector in a manner suitable for expressing
said protein;
c) transforming or otherwise introducing
said vector into an appropriate eucaryotic cr
prokaryctic host: cell forming a recombinant
host cell,
d) culturing said recombinant host cell in
a manner amenable to expression of said
protein; and
e) recovering and substantially purifying
said protein by any suitable means.
Expressing Recombinant BLT Fusion Protein in Procaryotic and
Eucaryotic Host Cells
Human beta-lipotropin (BLT) is an 89-amino acid hormone
that is produced by the pituitary in the form of a precursor
protein that undergoes post-translational processing to
generate several bioactive hormones including BLT. Because
BLT is small and contains proteolytically sensitive sines,
we have produced this protein initially by chemical
synthesis. However, this method is not useful for generating
large quantities. In this invention, we describe a method by
which BLT can be produced in bacterial or fungal expression
hosts in the form of fusion proteins. These fusion proreins
are protected from proteolytic degradation and allow for
recovery of intact BLT by methods described below.
In E. coli, 3LT is produced as a fusion protein w.-ich
can be recovered from cell lysates in the presence of high
salt by conventional purification methods. The fusion
protein contains a recognition site for specific proteases
which are used to separate the fusion partner from BLT. In
Pichia pastoris, the fusion protein is cleaved upon
secretion frcm the cell in such a way that native BLT can be
detected ana recovered intact from the culture medium.
This method offers a production process for 3LT.
Unlike chemical synthesis, the process described herein can
be scaled up to produce large amounts of BLT.
Procaryotes may be employed in the production of
recomoinant BLT protein. For example, the Escherichia coli
K12 strain 294 (ATCC No. 31446) is particularly useful for
expression of foreign proteins in a procaryotic cell. Other
strains of E. coli, bacilli such as Bacillus subtilis,
enterobacteriaceae such as Salmonella typhimurium or
Serratia marcescans, various Pseudomonas species and other
bacteria, such as Strepto/nyces., may also be employed as host
cells in the cloning and expression of the recombinant
proteins of this invention.
Promoter sequences suitable for driving the
expression of genes in procaryotes include b -lactamase
[e.g. vector pGX2907, ATCC 33344, contains a replicon and b
-lactamase gene], lactose systems [Chang et al., Nature
(London), 275:615 (1978); Goeddel et al., Nature (London),
281:544 (1S79)], alkaline phosphatase, and the tryptophan
(trp} promoter system [vector pATKl (ATCC 376S5)], which is
designed to facilitate expression of an open reading frame
as a trpE fusion protein under the control of the trp
promoter. Hybrid promoters such as the tac promoter
(isoiatable from plasnid pDR540, ATCC-37282) are also
suitable, as are T7 promoters. Still other bacterial
promoters, whose nucleotide sequences are generally known,
may be ligated to DNA encoding the protein of the instant
invention, using linkers or adapters to supply any required
restriction sites. Promoters for use in bacterial systems
also will contain a Shine-Dalgarno sequence operably-linked
to the DNA encoding the desired polypeptides. These
examples are illustrative rather than limiting.
The proteins of this invention may be synthesized
either by direct expression or as a fusion protein from
which the fusion partner may be removed by enzymatic or
chemical cleavage. In principle, this invention applies to
any fusion system that can be expressed in bacterial or
fungal hosts. In one embodiment: of a suitable fusion system,
a recognition site is placed between BLT and a fusion
partner, wherein, for example, the fusion partner is placed
at the amino terminal end of 3LT. A suitable site can be a
recognition sequence for a protease, or a site that is
susceptible to chemical cleavage.
Examples of suitable bacterial fusion partners include
glutathione-S-transferase, maltose binding protein,
procarboxypeptidase, calmodulin binding protein or any
amino-terminally located sequence that promotes high-level
expression of a fusion protein.
Examples of suitable proteases include factor Xa,
thrombin and enterokinase. Agents for chemical cleavage
include cyanogen bromide, acid, etc.
Examples of fusion partners that are useful in fungal
systems include the alpha mating factor, propeptide, human
serum albumin and any other sequence that promotes
expression and secretion of the fusion protein and
subsequent cleavage (during secretion) to release native BLT
into the culture medium.
In one approach, a BLT fusion protein comprises a
native 3LT sequence fused to a dipeptide (viz. Met-Arg> at
the amino terminal end of the native BLT molecule (e.g. SEQ
ID NO:2). The dipeptide partner of this fusion molecule can
be released by treatment with Cathepsin C, and the native
BLT molecule purified by techniques known in the art, such
as, for example, HPLC. In another method for producing
recombinant BLT fusion protein, glutathione-S-transferase
(GST) is used as the fusion partner to produce the protein
designated herein as SEQ ID NO:7, essentially as described
in Smith and Johnson (Gene, 67, 31, 1988), hereby
incorporated by reference.
It is often observed in the production of certain
peptides in recombinant systems that expression as a fusion
protein prolongs the lifespan, increases the yield of the
desired peptide, or provides a convenient means of purifying
the protein. This is particularly relevant when expressing
mammalian proteins in procaryotic hosts. A variety of
peptidases (e.g. enterokinase and thrombin) which cleave a
polypeptide at specific sites or digest the peptides frorr.
the amino or carboxy termini (e.g. diaminopeptidase) of the
peptide chain are known. Furthermore, particular chemicals
(e.g. cyanogen brcmide) will cleave a polypeptide chain at
specific sites. The skilled artisan will appreciate the
modifications necessary to the amino acid sequence (and
synthetic or semi-synthetic coding sequence if recombinant
means are employed) to incorporate site-specific internal
cleavage sites. See e.g., P. Carter, "Site Specific
Proteolysis of Fusion Proteins", Chapter 13, in Protein
Purification: From Molecular Mechanisms to Larae Scale
Processes, American Chemical Society, Washington, D.C.
(1990).
In addition to procaryotes, a variety of amphibian
expression systems such as frog oocytes, and mammalian cell
systems can be used. The choice of a particular host cell
depends to some extent on the particular expression vector
used. Exemplary mammalian host cells suitable for use in
the present invention include HepG-2 (ATCC HB 8065), CV-1
(ATCC CCL 70), LC-MK2 (ATCC CCL 7.1), 3T3 (ATCC CCL 92;,
CHO-K1 (ATCC CCL 61), HeLa (ATCC CCL 2), RPMI8226 (ATCC CCL
155), H4IIEC3 (ATCC CCL 1600), C127I (ATCC CCL 1616), KS-
Sultan (ATCC CCL 1484), and BHK-21 (ATCC CCL 10), for
example.
A wide variety of vectors are suitable for
transforming mammalian host cells. For example, the pSV2-
type vectors comprise segments of the simian virus 40 (SV4 0)
genome required for transcription and polyadenylation. A
large number of plasmid pSV2-type vectors have been
constructed, such as pSV2-gpt, pSV2-neo, p3V2-dhfr, pSV2-
hyg, and pSV2-b-globin, in which the SV40 promoter drives
transcription of an inserted gene. These vectors are widely
available from sources such as the American Type Culture
Collection (ATCC), 12301 Parklawn Drive, Rockville,
Maryland, 20852, or the Northern Regional Research
Laboratory (NRRL), 1815 N. University Street, Peoria,
Illinois, 61504.
Promoters suitable for expression in mammalian
cells include the SV4 0 late promoter, promoters from
eukaryotic genes, such as, for example, the estrogen-
indue ible chicken ovalbumin gene, the interferon genes, the
glucocorcicoid-inducible tyrosine aminotransferase gene, the
thymidine kinase gene promoter, and the promoters of the
major early and late adenovirus genes.
Transfection of mammalian cells with vectors can
be performed by a plurality of well known processes
including, but not limited to, protoplast fusion, calcium
phosphate co-precipitation, electroporation and the like.
See, e.g., Maniatis et al., supra.
Some viruses also make appropriate vectors.
Examples include the adenoviruses., the adeno-associated
viruses, the vaccinia virus, the herpes viruses, the
baculoviruses, and the rous sarcoma virus, as described in
U.S. Patent 4,775,624, incorporated herein by reference.
Eucaryotic microorganisms such as yeast and other
fungi are also suitable host cells. The yeasts
Saccharomyces cerevisiae and Pichia pastoris are the
preferred eucaryotic microorganisms. Other yeasts such as
Xluyveromyces lactis are also suitable. For expression in
Saccharomyces, the plasmid YRp7 (ATCC-40053), for example,
may be used. See, e.g., L. Stinchcomb et al., Nature, 282,
39 (1979); J. Xingsman et al., Gene, 7, 141 (1979); S.
Tschemper et al., Gene, 10, 157 (1980). Plasmid YRp7
contains the TRP1 gene which provides a selectable marker
for use in a trpl auxotrophic mutant.
Other embodiments of the present invention
comprise isolated nucleic acid sequences that encode BLT, cr
fragment thereof. As skilled artisans will recognize, the
amine acid compounds of the invention can be encoded by a
multitude of different nucleic acid sequences. Because these
alternative nucleic acid sequences would encode the same
amino acid sequences, the present invention further
comprises these alternate nucleic acid sequences.
The BLT encoding nucleic acids of the invention
may also be produced by chemical synthetic methods. The
synthesis of nucleic acids is well known in the art. See,
e.g., E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana,
Methods in Enzymology, 68:109-151 (1979) . Fragments of the
DNA sequence corresponding to BLT could be generated using a
conventional DNA synthesizing apparatus, such as the Applied
3iosystems Model 380A or 38OB DNA synthesizers (Applied
Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA
944 04) using phosphoramidite chemistry, thereafter ligating
the fragments so as to reconstitute the entire gene.
Alternatively, phosphotriester chemistry may be employed to
synthesize the nucleic acids of this invention. (See, e.g.,
M.J. Gait, ed.r Oligonucleotide Synthesis, A Practical
Approach, (1984)).
Vectors
Another aspect of the present invention relates to
recombinant DNA cloning vectors and expression vectors
comprising the nucleic acids of the present invention. The
preferred nucleic acid vectors are those which comprise DNA.
The most preferred recombinant DNA vectors comprise the
isolated DNA sequence, SEQ ID NO:l.
The skilled artisan understands that choosing the
most appropriate cloning vector or expression vector depends
uoon a number of factors including the availability of
restriction enzyme sites, the type of host cell into which
the vector is to be transfected or transformed, the purpose
of the transfection or transformation (e.g., stable
transformation as an extrachromosomal element, or
integration into the host chromosome) , the presence or
absence of readily assayable or selectable markers (e.g.,
antibiotic resistance and metabolic markers of one type and
another), and the number of copies of the gene desired in
the host cell.
Vectors suitable to carry the nucleic acids of the
present invention comprise RNA viruses, DNA viruses, lytic
bacteriophages, iysogenic bacteriophages, stable
bacteriophages, plasmids, viroids, and the like. The most
preferred vectors are plasmids.
When preparing an expression vector the skilled
artisan understands that there are many variables to be
considered, for example, whether to use a constitutive or
inducible promoter. The practitioner also understands that
the amount of nucleic acid or protein to be produced
dictates, in part, the selection of the expression system.
Regarding promoter sequences, inducible promoters are
preferred because they enable high level, regulatable
expression of an operably-linked gene. The skilled artisan
will recognize a number of suitable promoters that respond
to a variety of inducers, for example, carbon source, ir.etal
ions, and heat. Other relevant considerations regarding an
expression vector include whether to include sequences for
directing the localization of a recombinant protein. For
example, a sequence encoding a signal peptide preceding the
coding region of a gene is useful for directing Cne extra-
cellular export of a resulting polypeptide.
The present invention also provides a method for
constructing a recombinant host cell capable of expressing
BLT proteins, said method comprising transforming or
otherwise introducing into a host cell a recombinant DNA
vector that comprises an isolated DNA sequence that encodes
BLT. The preferred host cell is any cell that can accomodate
high level expression of an exogenously introduced gene or
protein. Transformed host cells may be cultured under
conditions well known to skilled artisans such that
recombinant BLT is expressed.
The present invention also provides methods for
treating humans and other mammals afflicted with diseases or
conditions associated with defects in insulin metabolism or
carbohydrate metabolism, such as, for example,
hyperglycemia, hyperinsulinemia, type 1 and type 2 diabetes,
and complications associated therewith. The method comprises
administering to an organism in need thereof an effective
amount of BLT protein or peptide, or fusion protein
comprising 3LT, or functional fragment thereof, or analog
thereof in a dose between about 1 and 1000 ug/kg body
weight. In practicing this method, BLT can be administered
in a single daily dose, in multiple doses per day, or by
continuous or discontinuous administration via a mechanical
pump device that is implanted in the body or attached
otherwise thereto. The amount of BLT or related protein or
peptide to be administered will be determined by the
physician, and depend on such factors as the nature and
severity of the condition, syndrome, or disease being
treated, and the age and general health of the patient.
The present invention also provides a
pharmaceutical composition comprising as the active agent a
BLT protein or functional fragment thereof, or analog
thereof e.g. that represented by SEQ ID NO:8, or a
pharmaceutically acceptable non-toxic salt thereof, in
admixture with a pharmaceutically acceptable solid or liquid
carrier. The protein, preferably in the form of a
pharmaceutically acceptable salt, can be formulated for
parenteral administration for the therapeutic or
prophylactic treatment of diabetes or complication thereof.
For example, compounds of SEQ ID NO:8 or other BLT, or
fragment thereof, can be admixed with conventional
pharmaceutical carriers and excipients. The compositions
comprising BLT protein contain from about 0.1 to 90% by
weight of the protein, preferably in a soluble form, and
more generally from about 10 to 30%. Furthermore, the
present proteins may be administered alone or in combination
with other anti-diabetic agents or agents useful in treating
diabetes.
For intravenous (IV) use, the BLT protein,
fragment, or analog is administered in commonly used
intravenous fluid(s) and administered by infusion. Such
fluids, for example, physiological saline. Ringer's solution
or 5% dextrose solution can be used.
For intramuscular preparations, a sterile
formulation, preferably a suitable soluble salt form of the
BLT protein, for example SEQ ID NO:8, such as the
hydrochloride salt, can be dissolved and administered z.r> a
pharmaceutical diluent such as pyrogen-free water
(distilled), physiological saline or 5% glucose solution. ~
suitable insoluble form of the compound may be prepared and
administered as a suspension in an aqueous base or a
pharmaceutically acceptable oil base, e.g. an ester of a
long chain fatty acid such as ethyl oleate.
The following examples more fully describe the
present invention and the use of BLT to treat diabetes.
Those skilled in the art will recognize that the particular
reagents, equipment, and procedures described are merely
illustrative and are not intended to limit the present
invention in any manner.
EXAMPLE 1
Solid Phase Synthesis and Purification of Human BLT Protein
A human BLT peptide (SEQ ID NO:8) was synthesized in a
single run using Fmoc chemistry. The synthesis of BLT is
complicated by the presence of several asp-gly dipeptiae
sequences in the N-terminal portion of the peptide. Aspartyi
side chains in asp-gly didpeptide sequences have been
observed to undergo base catalyzed cyclization and
subsequent addition with piperidine during FMOC synthesis.
This reaction is eliminated by use of Fmoc-(FmocKmb)-glycine
at each asp-gly sequence in the synthesis. Protection of the
glycyl amide with the Hmb group inhibits the cyclization of
the aspartyi side chain. Following cleavage, deprotection,
and reverse phase HPLC purification, the peptide can be
analyzed by electrospray mass spectrometry. The major
species seen in the synthesis is the full length peptide
having the expected mass. Use of this method allows
production of quantities of purified protein in excess of
100 mg from a single run at the 0.1 mmole scale.
Materials: Preloaded Frnoc-Glu (OtBu) Wang (1% cross-
linked polystyrene functionalized with p-benzoxybenzyl
alchohol) resin at approximately 0.6 mmole amino acid/g
resin. N-methylpyrrolidone (NMP), piperidine, 2-(lH-
benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTD), 2M N,N-Diisopopylethylamine
(DIEA) 1-hydroxybenzotriazole (HOBt), Dichloromethane (DCM),
Dimethylformamide (DMF).
Resin, preloaded with the Fmoc-protected C-terminal
amino acid (0.1 or 0.25 mmole animo acid), was weighed and
placed into a reaction chamber. The resin was preswollen by
washing with DCM. The resin was then washed with NMP.
The N-terminal Fmoc group was removed by incubation of the
resin in a 18-22 % solution of piperidine in NMP. Following
deprotection, the resin was extensively washed with NMP.
One mmole of the next Fmoc-protected amino acid to be
added to the peptide was solufcilized in 2.1 g of NMP, 2.0 g
of 0.45 M HBTU/HOBt reagent in DMF. Following the
solubilization, activation of the amino acid was initiated
by addition of 3 ml of 2M DIEA in NMP. The activated amino
acid was then added to the deprotected resin and allowed to
couple. Upon completion of coupling, the resin was washed
extensively with NMP. The complete cycle of deprotection and
coupling was then repeated for each successive amino acid.
Specific cycle steps in the synthesis were as follows:
Cycle AA Steps
For amino acids that react slowly or inefficiently, 2
separate coupling reactions were performed. Any residual
unreacted peptide was blocked by treatment with acetic
anhydride. The sequence of steps for one of these amino
acids was deprotection, coupling reaction 1, wash, coupling
reaction 2, wash, Ac20 cap, wash, deprotection.
Abreviations: OtBu: t-butyl ester, tBu: t-butyl, Boc:
t-butoxycarbonyl, Pmc: 2,2,5,7,8-pentaraethylchroma-6-
sulfonyl, Hmb: 2-hydroxy-4-methoxybenzyl, Fmoc: 9-
f luorenylmethoxycarbonyl.
EXAMPLE 2
Construction of a synthetic gene encoding BLT
Plasmid constructions were performed using standard
cloning methodologies as described by Maniatis et.al.,
Molecular Cloning: A laboratory Manual, Cold Spring Harbor
Laboratory, N.Y. (1982) . Emzymes and other reagents were
obtained from Gibco-BRL, Gaithersgurg, MD or New England
Biolabs, Beverly MA 01915. Methods for digesting,
identifying, recovering and purifying the various
oligonucleotides and DNA fragments used in this invention-
are known to those skilled in the art', as are methods for
ligation of these sequences into vectors, transforming host
microorganisms, and culturing these host microorganisms
under conditions allowing production of plasmid DNA and
recombinant protein products.
A DNA sequence encoding human BLT (SEQ ID NO:8) was
assembled from 8 chemically synthesized oligonucleotides
ranging in size from 52 to 86 bases. The oligonucleotides
were generated using a conventional DNA synthesizing
apparatus such as the Applied Biosystems model 380A or 380B
(commercially available from Applied Biosystems, Inc., 850
Lincoln Center Drive, Foster City, CA 94404). The sequence
of the oligonucleotides was designed in such a way that 4 of
the oligonucleotides generated one strand of the synthetic
gene and the remaining 4 oligonucleotides generated the
complementary strand of the synthetic gene. Prior to
assembly, the oligonucleotides were treated with
polynucleotide kinase in the presence of ATP to
phosphorylate the free hydroxyl groups of the individual
oligonucleotides.
The 8 oligonucleotides were mixed in eguimolar
concentration (3 picomoles/ul) in a volume of 100 ul, heated
to 95°C, and gradually cooled ~o room temperature over a
period of several hours. This allowed for proper base-
pairing of the individual oligonucleotides. T4 DNA ligase
was added to ligate the oligonucleotides and generate a
double-stranded -285 base-pair DNA, which was analyzed and
purified on a 2% agarose gel. This fragment, having "sticky"
ends, was ligated into a pUC18 vector digested with Ndel and
BamHI. The ligation mix was transformed into competent BH10B
cells which were plated onto tryptone-yeast agar plates
containing lOOug/ml of ampicillin. Colonies that grew
overnight were analyzed for the presence of plasmids that
contained the chemically synthesized gene. This was done by
purifying plasmid DNA from these colonies, digesting the
plasmid DNA with Ndel and BamH" and verifying the presence
of a -235 base-pair fragment. The correct sequence was
subsequently confirmed by DNA sequence analysis. The plasmid
was named pOJ83 8.
EXAMPLE 3
Construction of plasmids expressing a 3LT fusion protein
Because BLT is a small protein, it was advantageous to
increase its size by creating a fusion protein. For this
purpose, two different fusion partners were used. One was
glutathione-S-transferase (GST), having a factor Xa cleavage
site and the amino acid sequence designated herein as SEQ ID
NO:6, as encoded by vector pGEX-2T (Pharmacia
Biotechnology, Piscataway, NJ 08855); the other was a short
peptide sequence MIIGLMVGGVSGSGSGSGDDDDP (SEQ ID NO:3).
To obtain native beta-lipotropin, fusion proteins are
treated either chemically, or by enzymatic digestion, to
disassociate beta-lipotropin from its fusion partner. This
is accomplished by inserting sites for enzymatic or chemical
cleavage between the fusion partner and beta-lipotropin. For
i
GST fusions, recognition sites for enterokinase or factor Xa
were chosen; for the small synchetic peptide fusion (SEQ ID
NO:3) , a proline was inserted, thereby enabling chemical
cleavage following acid treatment.
A plasmid encoding a GST fusion with a factor Xa
recognition sequence (IEGR) was constructed as follows.
Plasmid pOJ83 8 (pUC18 containing the synthetic beta-
lipotropin; See Example 4) was digested with Ndel and Nrul,
and the largest vector fragment was gel-purified. A
synthetic linker of the sequence 5'-
TATGAGATCTATCGAAGGTCGTGAGCTCACCGGTCAGCGTGTTCG-3' (SEQ ID
NO:4) and its complement, were mixed in equimolar amour.ts,
heated to 95°C and allowed to anneal by gradually lowering
the temperature to about 25°C. The annealed linker was
ligated to the vector fragment, and the ligation mix was
transformed into competent DH10B cells. Transformed cells
were plated onto tryptone-yeast agar plates containing
lOOug/ml cf ampicillin. Colonies that grew overnight were
analyzed for the presence of plasmids that contained the
linker sequence. This was done by purifying plasmid DNA from
these colonies and verifying the presence of a new Bgill
site that was introduced via the linker. The correct linker
sequence was subsequently confirmed by DNA sequence
analysis. The resulting plasmid was named pOJ839.
Plasmid pOJ83 9 was digested with Bglll and EcoRI and
the small fragment containing the beta-lipotropin gene (now
attached to a factor Xa recognition sequence) was gel-
purified and ligated into pGEX 2-T digested with 3amHI and
EcoRI. The ligation mix was transformed into competent DH10B
cells, which were plated onto tryptone-yeast agar plates
containing lOOug/ml of ampicillin. Colonies that grew
overnight were analyzed for the presence of plasmids that
contained a new ~3 0 0 bp fragment following digestion with
Sad. This new plasmid was named pOJ840.
EXAMPLE 4
Recombinant Vector pHMM260-ProCpB(10)/LVPR/Beta lipotropin
(R49) Encoding Procarboxypeptidase-BLT Fusion Protein
A recombinant DNA expression vector, pHMM260, contains
a ProCpB(10)/LVPR/Beta Lipotropin (R49) expression cassette,
encoding a procarboxypeptidase-3LT fusion protein. This
vector was produced using standard cloning techniques. An
annotation of the expression cassette of pHMM260 is shown in
Figure 16. The expression cassette is flanked by an Ndel
site at the 5' end and a BamHI site at the 3' end. The pro-
peptide portion of porcine Procarboxypeptidase B protein
occurs at the 5' end of the cassette, beginning with a Met
residue and ending with a Ser residue. A thrombin protease
recognition sequence, LVPR, occurs immediately 3' of the
procarboxypeptidase sequence. The sequence beginning with a
Glu residue to the 3' end of the thrombin cleavage site
encodes wild-type human beta-lipotropin.
Several derivative cassettes were constructed such
that position 49 of the BLT sequence was changed from the
wild type sequence Arg (R49) to Ala (A49) pHMM261, or Glu
(E49) pHMM262, or Gin (Q49) pHMM263. These derivative were
made in order to reduce susceptibility to proteolysis during
synthesis and purification of the fusion proteins. All of
these vectors encode soluble fusion proteins.
EXAMPLE 5
Recombinant Vector pHMM268 Encoding Serine Hydroxy
Methyltransferase(SHMT)-BLT Fusion Protein
A fusion protein containing an amino-terminal sequence
from the enzyme serine hydroxy methylase transferase (SHMT)
was constructed using standard cloning techniques. The
plasmid pHMM268 was expressed at high levels in E. coli. The
fusion protein produced by pHMM268 was insoluble and could
be recovered with the particulate fraction. This feature
provided advantage in protection against proteolytic
degradation.
EXAMPLE 6
Administration of Mouse BLT for 7 Days to Male Avy/A Mice
The A^7'-1 mouse is a model for obesity and diabetes.
This inbred strain develops hyperglycemia, hyperinsulinemia,
and insulin insensitivity in mid-life, and therefore
provides a useful model for both obesity and diabetes.
Male A'77* mice were purchased from Harlan Co.
(Indianapolis, IN) at about 6 months of age. The animals
were housed, 6 per cage, and fed ad libitum 5008 feed and
water. Blood- glucose levels were measured at regular
intervals; when morning glucose levels reached at least
3 00mg/dL the animals were subjected to the experimental
protocol. Five animals, randomly selected, were housed
together as a control; 5 animals, randomly selected, were
housed together to receive treatment with mouse BLT (the
mouse BLT sequence is readily available; See e.g. M. Notake
et. al. FEBS Lett. 156, 67-71, 1983; herein incorporated by
reference), synthesized by solid phase techniques.
Mice undergoing treatment with BLT received 60jjg bid
subcutaneously (SC) (200ul total volume for each dose);
control animals received 200ul vehicle bid(saline solution).
The animals were injected twice daily, in the morning and in
the afternoon, for 6 days; on day 7 they received just the
morning injection.
Blood glucose levels, body weight, and food consumption
were measured in the morning on day 0. Body weight and food
consumption were measured in the morning on days 1 through
6. Plasma triglycerides were measured in the morning of day
5. Mice were fasted overnight from the afternoon of day 6
to the morning of day 7. On day 7, an oral glucose
tolerance test was performed. Zero time blood glucose
levels were measured 2 hours after the morning injection of
either saline or BLT. For this test, animals were
administered oral 25% dextrose solution (lOOul/lOg body
weight), then bled 30, 60, and 120 minutes later. The hlood
samples were used for determining levels of glucose and
insulin. After the oral glucose tolerance test, the animals
were fed ad libitum. On days 1, 3, and 14 after the last: SC
injection, of either saline or BLT, animals were bled to
determine blood glucose values.
RESULTS:
BLT Administration Lowers Blood Glucose and Insulin In Vivo
The results of these experiments are summarized in
Figures 1-8. Throughout the course of the 7 day treatment,
animals in the control group and the test group consumed
roughly 4.5 to 6 grains of food (Figure 1) . Body weight in
both groups remained the same in both groups, at about 50
grams (Figure 2).
BLT treatment decreased plasma triglycerides in treated
mice after 6 days of treatment (see Figure 3). The control
animals exhibited a mean value of 3.05 nmol/L (s.d. 0.58),
whereas treated animals were measured at a mean value of
2.05 nmol/L (s.d. 0.43). This represents a decline in serum
triglycerides of about 30% to 35%.
BLT also induced a substantial decline in blood glucose
levels in treated animals (Figure 4). After 7 days of
administration of BLT the treated group manifested blood
glucose levels of 135 mg/dL (s.d. 16); in marked contrast
the control group measured at 171 mg/dL (s.d. 16).
The decline in blood glucose levels in treated animals
continued for at least 3 days after the last administration
of BLT on day 7 (Figure 5). The control animals exhibited
glucose levels of 331 mg/dL (s.d. 79) . On the other hand,
treated animals maintained substantially lower blood glucose
levels, at 205 mg/dL (s.d. 76). The effect of BLT in
lowering blood glucose levels was not observed at 14 days
after the last administration of BLT (Figure 6).
To address the effects of BLT treatment on insulin
levels, an oral glucose tolerance test was performed after
the 7 day treatment period and an overnight fast (Figure 7
and Figure 8). Control and treated animals showed an initial
rise in blood glucose levels from about 80 mg/dL to 2 00
mg/dL, or greater, at the 3 0 minute time point after
initiation of the test; thereafter both groups showed a
comparable rate of decline in values, out to the 2 hour time
point (Figure 7). Strikingly, the treated animals exhibited
plasma insulin values substantially below the control
animals (Figure 8). For example, at the 3 0 minute time
point, control animals had insulin levels at 11.5 ng/ml
(s.d. 1.9), whereas treated animals had insulin levels of
6.0 ng/ml (s.d. 3.0).
EXAMPLE 7
Administration of Mouse BLT for 14 Days to Male A"^'* Mice
Male A*7''* mice were housed, 6 per cage, and fed ad
libitum 5008 feed and water. Blocd glucose levels were
measured at: regular intervals; when morning glucose levels
reached at least 3 00mg/dL the animals were subjected to the
experimental protocol. Five animals, randomly selected, were
housed together as a control; 5 animals, randomly selected,
were housed together and treated with mouse 3LT, which had
been synthesized by solid phase techniques.
Mice undergoing treatment with BLT received 6 0ug bid SC
(2 00ul total volume for each dose) ; control animals received
200ul vehicle (saline solution). The animals were injected
twice daily, in the morning and in the afternoon, for 13
days; on day 14 they received just the morning injection.
Blood glucose levels, body weight, and food consumption
were measured in the morning on day 0. Body weight and food
consumption were measured in the morning on days 1 through
13. Mice were fasted overnight from the afternoon of day 13
to the morning of day 14. On day 14, an oral glucose
tolerance test was performed- Zero time blood glucose
levels were measured 2 hours after the morning injection of
either saline or BLT. For this test, animals were
administered oral 25% dextrose solution (lOOul/lOg body
weight), then bled 30, 60, and 120 minutes later. Blood
samples were used for determining levels of glucose and
insulin. After the oral glucose tolerance test, the animals
were fed ad libitum. On days 1, 3, and 14 after the last SC
injection, of either saline or BLT, animals were bled to
determine blood glucose values.
RESULTS:
BLT Administration for 14 Days Lowers Blood Glucose and
Insulin
The results are summarized in Figures 9-10. Throughout
the 14 day treatment, animals in the control group and the
test group consumed roughly 4.5 to 6 grams of food and body
weights in both groups remained the same in both groupy, at
about 50 grams (data not shown).
To address the effects of a two week treatment wir.h BLT
on insulin levels, an oral glucose tolerance test was
performed after the 14 day period (Figure 9 and Figure 10).
Control and treated animals showed an initial rise in blood
glucose levels from about 80 mg/dL to 300 mg/dL, or greater,
at the 30 minute time point; thereafter both groups showed a
comparable race of decline in values, out to the endpoint of
the test at 2 hours (Figure 9). For example, treated animals
showed an average blood glucose level at the 3 0 min. time
point of 289 mg/dL (s.d. 24), whereas controls had levels of
348 mg/dL (s.d. 18). Lower levels were observed at the 60
min. and 12 0 min. time points also. For example, at 120 min.
treated animals had average blood glucose levels of 114
mg/dL (s.d. 4), whereas controls had levels of 188 mg/dL
(s.d. 23) .
Treated animals also exhibited plasma insulin values
substantially below the control animals (Figure 10). For
example, at the 3 0 minute time point, control animals had
insulin levels at 7.4 ng/ml (s.d. 2.1), whereas treated
animals had insulin levels of 4.8 ng/ml (s.d. 0.6).
EXAMPLE 8
BLT Stimulates Glucose Uptake in 3T3 Adipocytes
Mouse 3T3-L1 cells were plated in about 100 ul of
growth medium per well in 96 well plates such that about
25,000 cells were distributed per well.
Growth Medium: DMEM, high glucose, w/out L-glutamine
10% Calf serum
2mM L-glutamine
1% PenStrep
1.25ug/ml Fungizone
Cells were induced to differentiate into adipocytes 3 days
after plating by replacing growth medium with
Differentiation medium.
Differentiation medium: DMEM, high glucose, w/out L-
glutamine
10% FBS
2mM L-glutamine
1% Pen Strep
1.2 5ug/ml Fungizone
lOmM Hepes
0.25uM dexamethasone (lul/ml of
0.25mM stock)
0.5mM IBMX (lOul/ml of 50mM stock)
5ug/ml insulin (lul/ml 5mg/ml
stock)
Medium was removed from cells by aspiration using an 8
channel manifold attached to a house vacuum source with a
flow regulator. Fresh medium was dispensed into wells using
an 8 channel Matrix electronic pipettor, set to the slowest
speed, so as to minimize disruption of cells.
On day 1, lOOul Differentiation medium was added to
each well to initiate differentiation of 3T3 cells into
adipocytes. On day 3 after the start of differentiation, the
medium was changed to Differentiation medium containing
insulin, but without IBMX or dexamethasone (Insulin media).
On day 6, the medium was again changed to Differentiation
medium containing no insulin, IBMX, or dexamethasone (FBS
medium). Cells were maintained in FBS medium, with feeding
every other day, until ready for a glucose transport assay
from days 15 to 21 after the start of differentiation.
EXAMPLE 9
Glucose Transport Assay in 3T3 Cells Induced to
Differentiate into Adipocytes in the Presence of BLT
Mouse 3T3-L1 cells were induced to differentiate into
adipocytes as in Example 8. At 15 - 24 hours prior to
conducting a glucose transport assay, cells were treated as
follows:
1. Wells were washed twice with 100ml phosphate
buffered saline (PBS) at 37°C, aspirating between washes.
2. Next, 100ml DMEM, high glucose, 1%
Antibiotic/Antimycotic solution, 2mM glutamine, 0.1% BSA
(warmed to 37°C), 0 to 1000 nM mouse BLT (synthesized by
solid phase technique), and 0 to 10 0 nM insulin was added to
each well. This is the serum starvation phase.
3. Then, cells were incubated overnight at 37°C.
On the day of assay, cells were treated as follows:
1. Medium was removed, plates were blotted on paper
towels, and cells were washed twice with 100ml KRBH buffer
(Krebs-Ringer buffer containing Hepes, pK 7.4).
2. After removing the final wash, the cells were
incubated at 3 7°C for 1 hour in lOOul KRBK, 0.2% BSA with
lOOuM glucose, lOal/ml (0.luCi/well) radiolabeled 2-
Deoxyglucose (C14) , and desired insulin concentrations.
3. Following incubation with radiolabeled glucose, 10ul
of IGx cytochlasin 3 (200uM) was added to stop further
glucose uptake by cells.
4. Radiolabeled glucose uptake was determined by
reading plates on a Micrcbeta plate reader.
Results:
The results of this experiment are summarized in Figure
11. Glucose uptake into 3T3 cells induced to differentiate
into adipocytes was stimulated from about 3-fold to about 6-
fold when the adipocytes were pretreated prior to the uptake
assay with BLT.
EXAMPLE 10
Glucose Transport Assay in 3T3 Cells Induced to
Differentiate into Adipocytes in the Presence of Functional
Fragments of BLT
Mouse 3T3-L1 cells are induced to differentiate into
adipocytes as in Example 8. About 15 - 24 hours prior to
conducting a glucose transport assay, cells are treated as
follows:
1. Wells are washed twice with 100ml phosphate buffered
saline (PBS) at 37°C, aspirating between washes.
2. Next, 100ml DMEM, high glucose, 1%
Antibiotic/Antimycotic solution, 2mM glutamine, 0.1% BSA
(warmed to 3 7°C) , 0 to 100 0 nM mouse or human BLT
(synthesized by solid phase technique), and 0 to 100 nM
insulin is added to each well. This is the serum starvation
phase. For example, in one test human BLT fragment
designated herein as SEQ ID NO:10 is used; in another test,
human BLT fragment designated herein as SEQ ID NO:12 is
used.
3. Then, cells are incubated overnight at. 37°C.
On the day of assay, cells are treated as follows:
1. Medium is removed, plates are blotted on paper
towels, and cells are washed twice with 100ml KRBH buffer
(Krebs-Ringer buffer containing Hepes, pH 7.4).
2. After removing the final wash, the cells are
incubated at. 37°C for 1 hour in lOOul KRBH, 0.1% BSA with
lOOuM glucose, 10|il/ml (0. luCi/well) radiolabeled 2-
Deoxyglucose (C14) , and desired insulin concentrations.
3. Following incubation with radiolabeled glucose, lOul
of lOx cytochlasin B (200uM) is added to stop further
glucose uptake by cells.
4. Radiolabeled glucose uptake is determined by reading
plates on a Microbeta plate reader.
Results:
Glucose uptake into 3T3 cells induced to differentiate
into adipocytes is enhanced over the controls when
adipocytes are pretreated prior to the uptake assay with
functional fragments of BLT.
EXAMPLE 11
Functional Analogs of BLT
Mouse 3T3-L1 cells are induced to differentiate into
adipocytes as in Example 10. Adipocytes are then exposed to
analogs of human BLT, for example, SEQ ID NO:25 through SEQ
ID NO:35 and glucose transport is monitored as in Example 8.
Functional analogs exhibit substantially the same results as
native 3LT.
EXAMPLE 12
Administration of Human BLT for 14 Days to Male A^"'* Mice
Solid-phase synthesized human BLT was administered to
male A**''* mice in the dosage and according to the regimen
described in Example 7.
Results:
Human BLT administration lowered substantially serum
insulin levels in male Avy/A mice (See Figures 12 and 13) .
The results are summarized in Figures 12-13. Throughout
the 14 day treatment, animals in the control group and the
test group consumed roughly 4.5 to 6 grams of food and body
weights in both groups remained the same in both groups, at
about 50 grams (data not shown) ..
To address the effects of a two week treatment: with
human BLT on plasma glucose and insulin levels, an oral
glucose tolerance test was performed after the 14 day
period. Control animals and treated animals showed an
initial rise in blood glucose levels from about 80 mg/dL to
3 00 mg/dL, or greater, at the 3 0 minute time point;
thereafter both groups showed a comparable rate of decline
in values, out to the endpoint of the test at 2 hours
(Figure 12). For example, treated animals showed an average
blood glucose level at the 3 0 min. time point of about 4 00
mg/dL (s.d. 12), whereas controls had levels of 330 mg/dL
(s.d. 19). Lower levels were observed at the 60 min. and 120
min. time points also. For example, at 12 0 min. treated
animals had average blood glucose levels of 178 ng/dL (s.d.
16), whereas controls had levels of 202 mg/dL (s.d. 13).
Treated animals exhibited plasma insulin values
substantially below the control animals (Figure 13). Fcr
example, at the 3 0 minute time point, control animals had
insulin levels at 14.0 ng/ml (s.d. 1.5), whereas treated
animals had insulin levels of 10.2 ng/ml (s.d. 0.4). At the
120 minute time point, control animals had insulin levels at
9.3 ng/ml (s.d. 2.3) and treated animals had insulin levels
of 5.4 ng/ml (s.d. 0.9).
EXAMPLE 13
Mouse Beta Lipotropin Administration to Male Lep^/Lep0* Mice
Male Lep^/Lep^ mice purchased from Jackson
Laboratories (Bar Harbor, ME) were housed singly and fed ad
libitum 5008 feed and water. Mice were tail bled for initial
glucose, insulin, and triglyceride values. Mice were
selected for either control, 60fig, or I2 0ug bid mouse beta
lipotropin treatment. Treated mice received either 60^g bid
SC or 120ng bid SC (200jil total volume each dose) ; control
mice received 200ul vehicle (saline). Mice were injected at
7:00am and 3:00pm daily for 16 days, and injected at 7:00am
only on day 17. Body weight and food consumption were
measured in the morning on days 0 through 17. Mice were
tail bled the morning of day 7 for blood glucose, insulin,
and triglyceride values. The animals were fasted overnight
from the afternoon of day 13 to the morning of day 14. On
day 14, an oral glucose tolerance test was performed. Zero
time blood glucose and insulin were measured 2 hours after
the morning injection. Mice were administered oral 25%
dextrose solution (lOOul/lOg body weight), then bled 30, 60,
and 120 minutes later. Blood samples were used for
determining glucose and insulin. After the oral glucose
tolerance test, the mice were fed ad libitum. On day 15,
tail blood samples were taken to determine blood glucose,
insulin, triglyceride, and corticosterone values. On day
17, the mice were sacrificed.
The results from this experiment are shown in Figure
14. Plasma insulin values were significantly lowered in the
treated animals throughout the 16 day treatment period. For
example, at day 7 of the treatment the control animals
exhibited insulin values of about 300 ng/ml while treated
animals were measured at about 215 ng/ml, at least a 3 0%
diminution. Moreover, plasma insulin values remained lower
in treated animals at day 16 of the treatment (Figure 1A) .
EXAMPLE 14
Alzet™ Pump Administration of Human BLT to Male Lep^/Lep0"
Mice
Male Lepob/Lep^ mice purchased from Jackson
Laboratories(Bar Harbor, ME) at one month of age were housed
singly and fed ad libitum 5008 feed and water with lighting
on a 12 hour cycle (off from 6:00pm to 6:00am). Mice were
tail bled for initial glucose, insulin, and triglyceride
values. Treated animals received 0.24mg/day or 0.48mg/day
continuous administration of human beta lipotropin (ER4-VBH-
43) via subcutaniously implanted Alzet™ pumps (Alza, Inc.)
so that the average glucose and triglyceride values were
equal throughout ail 3 groups. Control animals were giver-
saline solution. The pumps were filled with saline for the
control group, and 2 0mg/ml human beta lipotropin for the low
dose group, and 40mg/ml human beta lipotropin for the high
dose group. Mice were anesthetized with isoflurane prior to
implantation of the pump. Body weight and food consumption
were measured on the morning of days 0 through 7. Mice were
tail bled the morning of day 4 for blood glucose, insulin,
and triglyceride values. Mice were fasted overnight froir. the
afternoon of day 6 to the morning of day 7.
On day 7, an oral glucose tolerance test was performed.
Mice were tail bled for 0 time insulin and glucose, then
administered oral 25% dextrose solution (lOOul/lOg body
weight), then bled 30, 60, and 120 minutes later. The blood
samples were used for determining glucose and insulin.
After the oral glucose tolerance test, the mice were fed ad
libitum.
The results of the glucose tolerance test respecting
Dlasma insulin levels are shown in Figure 15. The value at
0.24 mg BLT/day was about 25% lower than the control, while
the value at 0.48 mg BLT/day was about 40% lower than the
control (see Figure 15 inset for area under curves).
EXAMPLE 15
Solid Phase Synthesis and Purification of an analog of Human
3LT Protein
An analog of human BLT peptide (i.e. the analog
represented by SEQ ID NO:2o), in which Ala is substituted
for Glu in SEQ ID NO:8, was synthesized in a single run
using Fmoc chemistry. The synthesis is complicated by the
presence of several asp-gly dipeptide sequences in the N-
terminal portion of the peptide. Aspartyl side chains in
asp-gly didpeptide sequences have been observed to undergo
base catalyzed cyclization and subsequent addition with
piperidine during FMOC synthesis. This reaction is
eliminated by use of Fmoc-(FmocHmb)-glycine at each asp-gly
sequence in the synthesis. Protection of the glycyl amide
with the Hmb group inhibits the cyclization of the aspartyl
side chain. Following cleavage, deprotection, and reverse
phase HPLC purification, the peptide can be analyzed by
electrospray mass spectrometry. The major species seen in
the synthesis is the full length peptide having the expected
mass. Use of this method allows production of quantities of
purified protein in excess of 100 mg from a single run at
the 0.1 mmole scale.
Materials: Preloaded Fmoc-Glu (OtBu) Viang (1% cross-
linked polystyrene functionalized with p-benzoxybenzyl
alchohol) resin at approximately 0.5 mmole amino acid/g
resin. N-methylpyrrolidone (NMP), piperidine, 2-(1H-
benzotriazoi-l-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTD), 2M N,N-Diisopopylethylamine
(DIEA) 1-hydroxybenzotriazole (HOBt), Dichloromethane (DCM),
Dimethylformamide (DMF).
Resin, preloaded with the Fmoc-protected C-terminal
amino acid (0.1 or C.2 5 mmole animo acid), was weighed and
placed into a reaction chamber. The resin was preswollen by
washing with ECM. The resin was then washed with NMP.
The N-terminal Ftp.cc group was removed by incubation of the
resin in a 13-22 % solution of piperidine in NMP. Following
deprotection, the resin was extensively washed with NMP.
One mmole cf the next Fmoc-protected amino acid to be
added to the peptide was solubilized in 2.1 g of NMP, 2.0 g
of 0.45 K HBTU/H03t reagent in DMF. Following the
solubilization, activation of the amino acid was initiated
by addition of 3 ml of 2M DIEA in NMP. The activated amino
acid was then added to the deprotected resin and allowed to
couple. Upon completion of coupling, the resin was washed
extensively with NMP. The complete cycle of deprotectior. and
coupling was then repeated for each successive amino acid.
Specific cycle steps in the synthesis were as follows:
Cvcle AA Steps
For amino acids that react slowly or inefficiently, 2
separate coupling reactions were performed. Any residual
unreacted peptide was blocked by treatment with acetic
anhydride. The sequence of steps for one of these amino
acids was deprotection, coupling reaction 1, wash, coupling
reaction 2, wash, Ac20 cap, wash, deprotection.
Abreviations: OtBu: t-butyl ester, tBu: t-butyl, Boc:
t-butoxycarbonyl, Pmc: 2,2,5,7,8-pentamethylchroma-o-
sulfonyl, Hmb: 2-hydroxy-4-methoxybenzyl, Fmoc: 9-
fluorenylmethoxycarbonyl.
EXAMPLE 16
Alzet Pump Administration of Human BLT to Male ZD? Rats
Male Zucker Diabetic Fatty (ZDF) rats, purchased from
Genetic Models Inc. (Indianapolis, IN), were housed singly
and fed ad libitum 5008 feed and water. At five weeks of age
the rats were fasted overnight for an initial oral glucose
tolerance test. Blood was withdrawn from the tail for
determination of insulin and glucose levels at the 0 time
point, and then the animals were administered oral 50%
dextrose solution (2.5g/kg body wt.), and blood withdrawn
at 30, 60, and 120 minutes post-administration. After the
oral glucose tolerance test, the rats were fed ad libitum.
At 6 weeks of age, the animals were divided into groups of 4
animals each for administration of human BLT. On dav zero of
the test, tail blood was again withdrawn for the measurement
of glucose, insulin, corticosterone, and triglyceride
values. The rats were randomly selected for either control,
0.5mq/day, or 1.Omg/day continuous administration of human
beta lipotropin (ER4-VTA-2) via subcutaneously implanted
Alzet pumps. The pumps were filled with saline for
controls, 2 0.83 5mg/ml human beta lipotropin for the low dose
group, and 41.67mg/ml human beta lipotropin for the high
dose group. The pumps were incubated overnight at 37° in
saline prior to subcutaneous implantation. Body weight and
food consumption was measured on days 0 through 8. Blood
samples were taken the morning of day 3 for glucose,
insulin, corticosterone, and triglyceride values. Rats were
fasted overnight from the afternoon of day 5 to the morning
of day 6. On day 6, an oral glucose tolerance test was
performed. After the oral glucose tolerance test, the rats
were fed ad libitum.
The Zucker animals used in this study provide a good
model for type 2 diabetes. At 6 to 7 weeks of age blood
glucose values and triglyceride levels are rising while
insulin secretion is declining. These animals are
hypercorticosteronemic and insulin resistant.
The results presented in Table 3 and Figure 16
demonstrate a pronounced effect of human BLT on the ZDF rat
at a dose of 1 mg per day of human BLT. At 3 0 minutes after
the initiation of the OGTT plasma glucose levels were avg.
191 ± 16 mg/dL in the controls and 172 ± 5 mg/dL in the BLT-
treated animals (See Figure 16). At 60 minutes post-
initiation control animals displayed glucose levels of 157 +
6 mg/dL, while 3LT-treated animals were at 154 ± 8 mg/dL in
the BLT-treated animals.
Table 3 illustrates that blood glucose values remained
lower in BLT-treated animals than in control animals,
especially at the higher dose. The control animals displaed
glucose values of about 150 mg/dL at 0, 2, and 7 days
following the start of administration of BLT. Gn the other
hand animals treated with 1 mg/day of human BLT averaged
about 140 mg/dL at day 7, some 7% lower than the control
animals. Treated animals also showed about 11% lower serum
triglyceride levels than control animals (Table 3),
demonstrating the insulinocropic effect of BLT treatmenc.
EXAMPLE 17
Human BLT Stimulates Glucose Transport in
ZDF Rat Skeletal Muscle
A study was carried out to determine the effects of
beta-iipotropin on glucose transport in skeletal muscle.
Zucker Diabetic Fatty (ZDF/Gmi™-fa/fa) male rats were
obtained from Genetic Models Inc. (Indianapolis, IN) . The
rats were maintained on Purina Formulab 5008 rat chow (Puina
Mills, Inc., St. Louis, MO) and housed in a light controlled
room with alternating 12 hour cycles of light and dark. Rats
were singly housed and given free access to food and water.
To determine glucose transport in muscle tissue, the
animals from Example 16 were anesthetized via an inter-
peritoneal injection of pentabarbitol sodium (6.5 mg/100 gm
body weight), and the soleus muscles isolated and divided in
half. Each half of the tissue sample was washed in saline
for 2 minutes, and then in gassed (95% 02-5% C02) KHB with 1%
BSA, 8 mM glucose, and 32 mM mannitol.
Glucose transport assays were carried out as follows.
Pre-incubation:
Muscle tissue samples were transferred to 2 0 ml vials
containing 1.8 ml gassed KHB with 1% BSA, 8 mM glucose, 32
mM mannitol, with or without 500 nM mouse beta-lipotropin.
Samples were placed in a shaking waterbath for 2 0 hours at
room temperature with two intermittent buffer changes.
After pre-incubation, muscle tissue samples were washed
at 2S:C for 15 minutes under constant gassing of 95% 02-5%
CO-,. The samples were washed in vials contained 1.8 ml KHB
with 40 mM mannitol, insulin (2000 uU/ml) or no insulin, and
with or without beta-lipotropin (500 nM).
Muscle samples were then transferred to new vials under
constant gassing of 95% 02-5% C02. The incubation medium
consisted of 8 mM 3-0-Methyl-Glucose (OMG), 2 mCi/ml 3H-3 -
OMG, 3 0 mM mannitol, 0.3 mCi/ml 14C mannitol, 2mM
Pyruvate, insulin (2000 uU/ml), and mouse beta-lipotropin.
Controls lacked insulin and/or beta-lipotropin.
After 10 minutes at 29°C, samples were freeze clamped and
stored frozen until glucose transport could be assayed.
Glucose transport:
—
Muscles were trimmed to approximately 20-25 mg for digestion
in 0.5 ml 1M KOH at 70°C for 30 min. After digestion,
samples were placed on ice, and a 100 ul sample was removed
for glycogen analysis. To the remaining 0.4 ml sample, 0.4
ml of IN HCl was added and the tube vortexed. Then 3 00 ml of
the HCl-treated sample was added to 6.4 ml of scintillation
fluid and the radioactivity in the sample determined in a
scintillation counter.
Glucose transport was calculated on the basis of
intracellular 3H-3-OMG accumulation using I4C Mannitol as the
extracellular marker.
Glycogen levels:
To the 100 ui sample mentioned above 17 ul of glacial acetic
acid was added along with 500 ul 0.3 M Sodium acetate buffer
(?h 4.8+5 mg/mi amylogluccsidase). The tubes were then
incubated overnight at 37:C. The next day 50 ul of sample was
placed in a 96 well plate and 200 ul of Trinder reagent (Sigma
315-100; diluted to 20 ui with water) added. The plate was
WE CLAIM:
1. A substantially pure protein having an amino acid
sequence selected from the group consisting of SEQ ID NO:2,
3EQ ID NO:5, and SEQ ID NO:7.
2. An isolated nucleic acid compound encoding a protein c
Claim 1•
3. A vector comprising an isolated nucleic acid
compound of Claim 2.
4. A vector as in Claim 3, wherein said isolated
nucleic acid compound is operably-linked to a promoter
sequence.
5. A host cell containing a vector of Claim 4.
6. A method for constructing a recombinant: host cell
having the potential to express beta-lipotropin, said method
comprising introducing into said host cell by any suitable
means a vector of Claim 5.
7. A method for expressing beta-lipotropin in a
recombinant host cell of Claim 6, said method comprising
culturing said recombinant host cell under conditions
suitable for gene expression.
8. A pharmaceutical formulation comprising as an actzive
ingredient beta-lipotropin, analog, or functional fragment
thereof, associated with one or more pharmaceutical^
acceptable carriers, excipients, or diluents therefor.
9. A pharmaceutical formulation as in Claim 8 wherein
said beta-lipotropin is human beta-lipotropin.
10. Beta-lipotropin, analog, or functional fragment
thereof, for use in treating diabetes or complication
thereof.
11. A method for treating diabetes in a mammal
comprising administration of a therapeutically effective
amount of beta-lipotropin or analog thereof.
12. A method for treating diabetes in a mammal
comprising administration of a therapeutically effective
amount of a functional fragment of beta-lipotropin.
13. A method, as in Claim 11 wherein said beta-
lipotropin is human beta-lipotropin, having a sequence
identified herein as SEQ ID NO:8.
14. A method as in Claim 11 wherein said beta-
lipotropin is recombinant human beta-lipotropin.
15. A method as in Claim 11 wherein said diabetes is
Type 1 or Type 2 diabetes.
16. A method as in Claim 11 wherein said mammal is a
human.
17. A method for treating complications of diabetes in
a patient in need thereof comprising administering a
therapeutically effective amount of beta-lipotropin, analog,
or functional fragment thereof.
18. A method for lowering blood glucose levels in a
mammal by administering an effective amount of beta-
lipotropin, analog, or functional fragment thereof.
19. A method for treating hyperglycemia in a mammal in
need thereof by administering an effective amount of beta-
lipotropin, analog, or functional fragment thereof.
20. A method for treating hyperinsulinemia in a mammal
by administering an effective amount of beta-lipotropin,
analog, or functional fragment thereof.
21. A method for enhancing insulin sensitivity in a
mammal by administering an effective amount of beta-
lipotropin, analog, or functional fragment thereof.
22. A sclid-phase synthetic method for synthesizing
beta-lipctropin, analog, or fragment thereof, in a single
run, comprising the steps cf:
a) activating an amino acid having its oc-amino group,
and alternatively, side chain functional group, protected ~o
form a reactive ester;
b) attaching said activated amino acid to an inert
solid support;
c) activating a second protected amino acid to an ester
wherein the a-amino group of the first amino acid is de-
protected yielding a reactive amine, and the second
activated amino acid is reacted with said first amino acid
to form a di-peptide on the solid support;
d) repeating of steps (a) to (c);
e) removing the peptide from the solid support and
deprotecting side chain functional groups;
f) separating the peptide from the solid support; and
g) purifiying the peptide by any suitable means;
wherein,
h) where the BLT sequence contains a ser-pro-pro
tripeptide segment, multiple-coupling of the pro
residues is carried out;
i) after completion of the coupling step, any
unreacted, deprotected peptide is blocked to prevent,
chain lengthening of a deletion peptide; and
j) where the BLT sequence contains an asp-gly dipeptide
sequence, utilization of an N-a. protecting group at
each glycine residue preceding an asp residue.
23. A process for preparing beta-lipotropin as claimed
in Claim 1 comprising:
a. transforming a suitable host with an expression
vector wherein said vector encodes a beta-lipotropin;
b. culturing said transformed host under conditions
that enable expression of said beta-lipotropin;
c. purifying said beta-lipotropin by any suitable
means.
24. An assay for beta-lipotropin activity comprising
the steps of:
a) administering to a mammal that exhibits insulin
insensitivity and elevated blood glucose levels a test
protein; and
b) testing by any suitable means for blood glucose and
insulin levels after step (a) .
25. A method, as in Claim 11 wherein said beta
lipctropin is selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:7, and SEQ ID NO:8.
26. A peptide having insulinotropic activity selected
from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ
ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.
27. A peptide having insulinotropic activity selected
from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID
NO :24 and SEQ ID NO:25.
28. A peptide having insulinotropic activity selected
from the group consisting of SEQ ID NO: 9, SEQ ID NC:1C, SEQ
ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:13, SEQ ID
N0:19, SEQ ID N0:2C, SEQ ID N0:2i, SEQ ID NO:22, SEQ ID
NO:23, SEQ ID NO:24 and SEQ ID NO:25.
29. A method for treating diabetes comprising
administration of a therapeutically effective amount of at
least one peptide selected from the group consisting of SEQ
ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID
NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID
NO:25.
30. A method for treating diabetes comprising
administration of a therapeutically effective amount of at
least one peptide selected from the group consisting of SEQ
ID NO:9, SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, and SEQ
ID NO:13.
31. A pharmaceutical formulation comprising as an
active ingredient a peptide having insulinotropic activity
selected from the group consisting of SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID
NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
32. A functional analog of beta-lipctropin.
33. A fragment of beta-lipotropin having insuiinorropic
activity in vitro or in vivo.
34. A functional analog of beta-lipotropin that is
about 90% to 95% identical with SEQ ID NO:8, wherein amino
acid substitutions are selected from the group consisting
of :
the: residue at position l is alternatively Glu, Ala,
Asp or Gin;
the residue at position 2 is alternatively Leu, lie,
Val, or Met;
the residue at position 3 is alternatively Thr, Ala,
Glu, Ser, Pre, or Gly;
the residue at position 4 is alternatively Gly, Arg,
A-la, Leu, Pro, or Ser;
the residue at position 5 is alternatively Glu, Gin,
Asp, Asn, or Ala;
the residue at position 6 is alternatively Arg, Glu,
Leu, Lys, Gin, or Ala;
the residue at position 7 is alternatively Leu, Pro,
Asp, Val, lie, or Met;
the residue at position 8 is alternatively Arg, Glu,
Ala, Tyr, Leu, Lys, Pro, Gin, or Trp;
the residue at position S is alternatively Glu, Ala,
Pro, Asp, Asn, or Gin;
the residue at position 10 is alternatively Gly, Ala,
Ser, or Asp;
the residue at position 11 is alternatively Asp, Arg,
Pro, Asn, Gin, Ala, or Glu;
the residue at position 12 is alternatively Gly, Ala,
Ser, or Met;
the residue at position 13 is alternatively Pro, Glu,
Giy, or Val;
the residue at position 14 is alternatively Asp, Glu,
Asn, Gin, or Gly;
the residue at position 15 is alternatively Gly, Ala,
Ser, or Glu;
the residue at position 16 is alternatively Pro, Gin,
Leu, Gly, or Glu;
the residue at position 17 is alternatively Ala, Asp,
Ser, or Gly;
the residue at position 18 is alternatively Asp, Glu,
Gin, or Asn;
the residue at position 19 is alternatively Asp, Glu,
Asn, or Gin;
the residue at position 2 0 is alternatively Gly, Ser,
or Ala;
the residue at position 21 is alternatively Ala, Gly,
Ser, or Phe;
the residue at position 22 is alternatively Gly, Ala,
Ser, or Lys;
the residue at position 23 is alternatively Ala, Phe,
Thr, Gly, Ser, or Leu;
the residue at position 24 is alternatively Gin, Arg,
Asp, Asn, Leu, or Val;
the residue at position 25 is alternatively Ala, Leu,
Asp, lie, Gly, Ser, or Thr;
the residue at position 2 5 is alternatively Asp, Glu,
Gly, Asn, Gin, or Lys;
the residue at position 27 is alternatively Leu, Ala,
lie, Met, or Val;
the residue at position 2 8 is alternatively Glu, Gin,
Asn, or Asp;
the residue at position 29 is alternatively His, Asn,
Tyr, Ala, Gin or Glu;
the residue at position 30 is alternatively Ser, Gly,
Glu, Ala, Leu, or Asp;
the residue at position 31 is alternatively Leu, Ala,
Val, Met, or lie;
the residue at position 32 is alternatively Leu, Ala,
Val, lie, Met, or Pro;
the residue at position 33 is alternatively Val, Ala,
Glu, Leu, lie, Met, or Arg;
the residue at position 34 is alternatively Ala, Ser,
Pro, Glu, or Gly;
the residue at position 35 is alternatively Ala, Asp,
Gly, Ser, or Leu;
the residue at position 36 is alternatively Glu, Ala,
Thr, Leu, Asp, Asn, or Gin;
the residue at position 37 is alternatively Lys, Glu,
Thr, Arg, Gin, or Asp;
the residue at position 38 is alternatively Lys, Arg,
Gin, or Glu;
the residue at position 39 is alternatively Asp, Ala,
Asn, Glu, Gin, or Lys;
the residue at position 4 0 is alternatively Glu, Ser,
Asp, Asn, Gin, or Gly;
the residue at position 41 is alternatively Gly, Ala,
or Ser;
the residue at position 42 is alternatively Pro, Gly,
Ser, or Asn,-
the residue at position 43 is alternatively Tyr, Phe,
or Trp;
the residue at position 44 is alternatively Arg, Lys,
Gin, or Glu;
the residue at position 45 is alternatively Met, He,
Ser, or Val;
the residue at position 46 is alternatively Glu, Gin,
Asp, Asn, His, Arg or Gly;
the residue at position 4 8 is alternatively Tyr or Trp;
the residue at position 4 9 is alternatively Arg or Lys;
the residue at position 50 is alternatively Trp, Tyr,
or Phe;
the residue at position 51 is alternatively Gly, Ala,
Ser, or gin;
the residue at position 52 is alternatively Ser, Thr,
Asn, or Ala;
the residue at position 53 is alternatively Pro or Gly;
the residue at position 54 is alternatively Pro, Ala,
Gly, Arg, Leu, or Thr;
the residue at position 55 is alternatively Lys, Arg,
Gin, or Ala;
the residue at position 55 is alternatively Asp, Asn,
Glu, Gin, Ala, Gly, or Ile,-
the residue at position 57 is alternatively Lys, Gin,
or Arg;
the residue at position 58 is alternatively Arg, Gin,
or Lys;
the residue at position 59 is alternatively Tyr, Phe or
Trp,-
the residue at position 60 is alternatively Gly, Ala,
or Ser;
the residue at position 51 is alternatively Gly, Ala,
cr Ser;
the residue at position 52 is alternatively Phe, Tyr,
or Trp;
the residue at position 63 is alternatively Met, Leu,
lie, or Val;
the residue at position 64 is alternatively Thr, Ala,
Ser, or Lys;
the residue at position 65 :ls alternatively Ser, Ala,
Thr, or Pro;
the residue at position 66 is alternatively Glu, Asp,
Asn, Lys, or Gin;
the residue at position 57 is alternatively Lys, Arg,
or Gin;
the residue at position 58 is alternatively Ser, Ala,
Thr, or Gly;
the residue at position 69 is alternatively Gin, Glu,
Asp, Asn, Arg, or His;
the residue at position 70 is alternatively Thr.. Ser,
Ala, or Lys;
the residue at position 71 is alternatively Pro or Gly;
the residue at position 72 is alternatively Leu, lie,
Met, or Val;
the residue at position 73 is alcemacively Val, Leu,
lie, or Met;
the residue at position 74 is alternatively Thr, Ala,
or Ser;
the residue at position 75 is alternatively Leu, lie,
Me.t, or Val;
the residue at position 7 6 is alternatively Phe, Tyr,
Trp, or Leu;
the residue at position 77 is alternatively Lys, Gin,
or Arg;
the residue at position 78 is alternatively Asn, Asp,
Glu, Gin, or His;
the residue at position 79 is alternatively Ala, Gly,
Ser, lie or Val;
the residue at position 80 is alternatively lie. Leu,
Met, Val, or Thr;
the residue at position 81 is alternatively lie. Met,
Val, Thr, or Leu;
the residue at position 82 is alternatively Lys, Gin,
or Arg;
the residue at position 83 is alternatively Asn, Asp,
Glu, Gin, or Ser;
the residue at position 8-i is alternatively Ala, Val,
Ser, Gly, or Glu;
the residue at position 85 is alternatively Tyr, Phe,
Trp, or Kis;
the residue at position 85 is alternatively Lys, Gin,
or Arg;
the residue at position 8 7 is alternatively Lys, Gin,
or Arg;
the residue at position 3 3 is alternatively Gly, Ala,
cr Ser;
the residue at position 89 is alternatively Glu, Gin,
Asp, Asn, or His.
35. A functional analog of beta-lipotropin as in Claim
34 wherein said analog is about between 95% to 99% identical
with SEQ ID NO:8.
36. A peptide having insulinotropic activity selected
from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ
ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID
NO: 20, SEQ ID NO-.21, SEQ ID NO: 22.
37. A Method as in Claim 22, wherein at step:
h)where the BLT sequence contains a ser-pro-pro
tripeptide segment, double-coupling of the pro and ser
residues is carried out;
i) after completion of the coupling step, any
unreacted, deprotected peptide is blocked with an
anhydride to prevent chain lengthening of a deletion
peptide.
33. A method as in claim 37, wherein at step:
j) said N-cc protection comprising N-a-Hmb or methyl
benzoate.
39. A functional analog of beta-lipotropin that is at
least 90% identical with SEQ ID NO:8.

The present invention, provides isolated proteins
comprising beta-lipotropin (3LT), analogs thereof, fragments
thereof, nucleic acids encoding same, and methods for
producing and using BLT in the treatment of diabetes and
complications associated therewith.