U.S. patent application number 11/400715 was filed with the patent office on 2007-02-22 for differentiation of non-insulin producing cells into insulin producing cells by glp-1 or exendin-4 and uses thereof.
Invention is credited to Josephine Egan, Nigel Greig, Joel Habener, Harold Holloway, Antonino Passaniti, Riccardo Perfetti, Doris Stoffers.
Application Number | 20070041951 11/400715 |
Document ID | / |
Family ID | 22254191 |
Filed Date | 2007-02-22 |
United States Patent
Application |
20070041951 |
Kind Code |
A1 |
Egan; Josephine ; et
al. |
February 22, 2007 |
Differentiation of non-insulin producing cells into insulin
producing cells by GLP-1 or exendin-4 and uses thereof
Abstract
The present invention relates to a population of insulin
producing cells made by a process comprising contacting non-insulin
producing cells with a growth factor selected from the group
consisting of GLP-1 or Exendin-4, growth factors having amino acid
sequences substantially homologous to GLP-1 or Exendin-4, and
fragments thereof. The present invention also relates to methods of
differentiating non-insulin producing cells into insulin producing
cells and of enriching a population of cells for insulin-producing
cells. The present invention also relates to methods of treating
diabetes.
Inventors: |
Egan; Josephine; (Baltimore,
MD) ; Perfetti; Riccardo; (Washington, DC) ;
Passaniti; Antonino; (White Hall, MD) ; Greig;
Nigel; (Silver Spring, MD) ; Holloway; Harold;
(Middle River, MD) ; Habener; Joel; (Newton
Centre, MA) ; Stoffers; Doris; (Moorestown,
NJ) |
Correspondence
Address: |
NATIONAL INSTITUTE OF HEALTH;C/O NEEDLE & ROSENBERG, P.C.
SUITE 1000
999 PEACHTREE STREET
ATLANTA
GA
30303
US
|
Family ID: |
22254191 |
Appl. No.: |
11/400715 |
Filed: |
April 7, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09762538 |
Jul 19, 2001 |
7056734 |
|
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PCT/US99/18099 |
Aug 10, 1999 |
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11400715 |
Apr 7, 2006 |
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60095917 |
Aug 10, 1998 |
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Current U.S.
Class: |
424/93.7 ;
435/325; 435/366 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 3/10 20180101; A61P 5/50 20180101; A61K 38/26 20130101; C07K
14/57563 20130101; C12N 2501/335 20130101; C12N 5/0676 20130101;
A61K 35/39 20130101; A61P 5/48 20180101 |
Class at
Publication: |
424/093.7 ;
435/366; 435/325 |
International
Class: |
A61K 35/39 20070101
A61K035/39; C12N 5/08 20060101 C12N005/08 |
Claims
1-52. (canceled)
53. A method of differentiating non-insulin-producing cells into
insulin-producing cells in a subject with beta-cell dysfunction,
comprising contacting the non-insulin-producing cells for at least
two days with an amount of a substance effective to induce
differentiation of the non-insulin-producing cells by administering
the substance to the subject to provide said contact, wherein the
substance comprises a peptide selected from the group consisting of
exendin-4, an exendin-4 peptide, an exendin-4 peptide containing
one or more conservative amino acid substitutions at positions
other than positions 1, 4, 6, 7 and 9 of exendin-4, and a fragment
of any one of the preceding exendin-4 peptides, wherein the
exendin-4 peptide or fragment thereof has the ability to
differentiate non-insulin-producing cells into insulin-producing
cells, and wherein administration of the substance is discontinued
after insulin-producing cells are produced.
54. The method of claim 53, wherein the contacting is for at least
five days.
55. The method of claim 54, wherein the contacting is for at least
seven days.
56. The method of claim 55, wherein the substance is administered
subcutaneously.
57. The method of claim 55, wherein the subject has type I
diabetes.
58. The method of claim 55, wherein the subject has type II
diabetes.
59. The method of claim 55, wherein the substance is exendin-4.
60. The method of claim 55, wherein the non-insulin-producing cells
are human pancreatic cells.
61. The method of claim 55, wherein the administration of the
substance is discontinued for at least two weeks after the
insulin-producing cells are produced.
62. The method of claim 61, wherein the administration of the
substance is discontinued for at least two weeks after the
insulin-producing cells are produced and the subject's fasting
blood glucose is improved compared to that prior to administration
of the substance.
63. The method of claim 62, wherein the subject has type I
diabetes.
64. The method of claim 63, wherein the substance is exendin-4.
65. The method of claim 62, wherein the subject has type II
diabetes.
66. The method of claim 65, wherein the substance is exendin-4.
67. The method of claim 61, wherein the administration of the
substance is discontinued for at least two weeks after the
insulin-producing cells are produced and the subject's fasting
blood insulin level is improved compared to that prior to
administration of the substance.
68. The method of claim 67, wherein the subject has type I
diabetes.
69. The method of claim 68, wherein the substance is exendin-4.
70. The method of claim 67, wherein the subject has type II
diabetes.
71. The method of claim 70, wherein the substance is exendin-4.
72. The method of claim 53, wherein the substance is administered
at a dose of from about 1 pmole/kg to about 400 pmoles/kg.
73. The method of claim 72, wherein the substance is administered
at a dose of from about 10 pmoles/kg to about 40 pmoles/kg.
74. The method of claim 53, wherein the substance is administered
subcutaneously, intramuscularly, or intraperitoneally.
75. The method of claim 74, wherein the substance is administered
subcutaneously.
76. The method of claim 53, wherein the subject has type I
diabetes.
77. The method of claim 53, wherein the subject has type II
diabetes.
78. The method of claim 53, wherein the administration of the
substance is discontinued for at least 2 weeks after the
insulin-producing cells are produced.
79. The method of claim 53, wherein the substance is exendin-4.
80. The method of claim 53, wherein the substance is an exendin-4
peptide containing one or more conservative amino acid
substitutions at positions other than positions 1, 4, 6, 7 and 9 of
exendin-4.
81. The method of claim 53, wherein the substance is the fragment
of exendin-4, an exendin-4 peptide, or an exendin-4 peptide
containing one or more conservative amino acid substitutions at
positions other than 1, 4, 6, 7 and 9 of exendin-4.
82. The method of claim 53, wherein the non-insulin-producing cells
are human pancreatic cells.
83. The method of claim 82, wherein the human pancreatic cells are
acinar cells.
84. The method of claim 53, wherein the non-insulin-producing cells
are stem cells.
85. The method of claim 84, wherein the stem cells are human
pancreatic stem cells.
86. A method of differentiating non-insulin-producing cells into
insulin-producing cells in a subject with beta-cell dysfunction,
comprising contacting the non-insulin-producing cells for at least
two days with an amount of a substance effective to induce
differentiation of the non-insulin-producing cells by administering
the substance to the subject to provide said contact, wherein the
substance comprises a peptide selected from the group consisting of
exendin-4, an exendin-4 peptide, an exendin-4 peptide containing
one or more conservative amino acid substitutions at positions
other than positions 1, 4, 6, 7 and 9 of exendin-4, and a fragment
of any one of the preceding exendin-4 peptides, wherein the
exendin-4 peptide or fragment thereof has the ability to
differentiate non-insulin-producing cells into insulin-producing
cells, and wherein administration of the substance is discontinued
after the subject's fasting blood glucose is improved compared to
that prior to the administration of the substance.
87. The method of claim 86, wherein the contacting is for at least
seven days.
88. The method of claim 87, wherein the administration of the
substance is discontinued for at least two weeks after the
subject's fasting blood glucose is improved compared to that prior
to the administration of the substance.
89. The method of claim 88, wherein the substance is administered
subcutaneously.
90. The method of claim 88, wherein the subject has type I
diabetes.
91. The method of claim 90, wherein the substance is exendin-4.
92. The method of claim 88, wherein the subject has type II
diabetes.
93. The method of claim 92, wherein the substance is exendin-4.
94. The method of claim 88, wherein the non-insulin-producing cells
are human pancreatic cells.
95. The method of claim 88, wherein the substance is exendin-4.
96. The method of claim 88, wherein the substance is administered
at a dose of from about 10 pmoles/kg to about 40 pmoles/kg.
97. A method of differentiating non-insulin-producing cells into
insulin-producing cells in a subject with beta-cell dysfunction,
comprising contacting the non-insulin-producing cells for at least
two days with an amount of a substance effective to induce
differentiation of the non-insulin-producing cells by administering
the substance to the subject to provide said contact, wherein the
substance comprises a peptide selected from the group consisting of
exendin-4, an exendin-4 peptide, an exendin-4 peptide containing
one or more conservative amino acid substitutions at positions
other than positions 1, 4, 6, 7 and 9 of exendin-4, and a fragment
of any one of the preceding exendin-4 peptides, wherein the
exendin-4 peptide or fragment thereof has the ability to
differentiate non-insulin-producing cells into insulin-producing
cells, and wherein administration of the substance is discontinued
after the subject's fasting blood insulin level is improved
compared to that prior to the administration of the substance.
98. The method of claim 97, wherein the contacting is for at least
seven days.
99. The method of claim 98, wherein the administration of the
substance is discontinued for at least two weeks after the
subject's fasting blood insulin level is improved compared to that
prior to the administration of the substance.
100. The method of claim 99, wherein the substance is administered
subcutaneously.
101. The method of claim 99, wherein the subject has type I
diabetes.
102. The method of claim 101, wherein the substance is
exendin-4.
103. The method of claim 99, wherein the subject has type II
diabetes.
104. The method of claim 103, wherein the substance is
exendin-4.
105. The method of claim 99, wherein the non-insulin-producing
cells are human pancreatic cells.
106. The method of claim 99, wherein the substance is
exendin-4.
107. The method of claim 99, wherein the substance is administered
at a dose of from about 10 pmoles/kg to about 40 pmoles/kg.
108. A method of differentiating non-insulin-producing cells into
insulin-producing cells in a subject with beta-cell dysfunction,
comprising contacting the non-insulin-producing cells for at least
three days with an amount of a substance effective to induce
differentiation of the non-insulin-producing cells by administering
the substance to the subject to provide said contact, wherein the
substance comprises a peptide selected from the group consisting of
a GLP-1 peptide, a GLP-1 peptide containing one or more
conservative amino acid substitutions at positions other than
positions 7, 10, 12, 13 and 15 of GLP-1, and a fragment of any one
of the preceding GLP-1 peptides, and wherein the GLP-1 peptide or
fragment thereof has the ability to differentiate
non-insulin-producing cells into insulin-producing cells, and
wherein administration of the substance is discontinued after
insulin-producing cells are produced.
109. The method of claim 108, wherein the contacting is for at
least seven days.
110. The method of claim 109, wherein the substance is administered
subcutaneously.
111. The method of claim 109, wherein the subject has type I
diabetes.
112. The method of claim 109, wherein the subject has type II
diabetes.
113. The method of claim 109, wherein the non-insulin-producing
cells are human pancreatic cells.
114. The method of claim 109, wherein the administration of the
substance is discontinued for at least 2 weeks after the
insulin-producing cells are produced.
115. The method of claim 114, wherein the administration of the
substance is discontinued for at least 2 weeks after the
insulin-producing cells are produced and the subject's fasting
blood glucose or the subject's fasting blood insulin level is
improved compared to that prior to administration of the
substance.
116. The method of claim 115, wherein the subject has type I
diabetes.
117. The method of claim 115, wherein the subject has type II
diabetes.
118. The method of claim 109, wherein the substance is a GLP-1
peptide.
119. The method of claim 109, wherein the substance is a GLP-1
peptide containing one or more conservative amino acid
substitutions at positions other than positions 7, 10, 12, 13 and
15 of GLP-1.
120. The method of claim 109, wherein the substance is the fragment
of a GLP-1 peptide, or a GLP-1 peptide containing one or more
conservative amino acid substitutions at positions other than
positions 7, 10, 12, 13 and 15 of GLP-1.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/762,538, filed on Jul. 19, 2001 (now
allowed), which is a 35 U.S.C. .sctn. 371 application of
PCT/US99/18099, filed Aug. 10, 1999, which claims priority to U.S.
Application No. 60/095,917, filed Aug. 10, 1998. The aforementioned
applications are hereby incorporated herein in their entirety by
this reference.
[0002] The government has certain rights to this invention.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to a population of insulin
producing cells differentiated from non-insulin producing cells by
contacting the non-insulin producing cells with Glucagon-like
peptide-1 ("GLP-1"), exendin-4, or related peptides. The present
invention also relates to the methods for obtaining the insulin
producing cells and therapeutic uses in the treatment of diabetes
mellitus.
[0005] 2. Background Art
[0006] The mammalian pancreas is composed of two distinct types of
glandular tissue, the exocrine cells that secrete digestive enzymes
into the intestine and the endocrine cells that secrete hormones
into the blood stream. Endocrine cells were traditionally believed
to develop from the neural crest whereas the exocrine cells were
believed to develop from the endoderm. More recent work suggests
that these two cell types can come from common endodermal precursor
cells located along the epithelial lining of the ducts (Teitelman,
1996). It should be noted that the endocrine cells are terminally
differentiated and do not divide to make new endocrine cells.
Pancreatic endodermal precursor cells are the only cells thought to
produce new pancreatic endocrine cells.
[0007] The pancreas consists of ducts, which carry the exocrine
enzymes (amylase and lipase) to the intestine; acinar cells, which
produce the exocrine enzymes; and islets of Langerhans, which
contain the endocrine cells that produce and secrete insulin,
amylin, and glucagon. These hormones help to maintain normal blood
glucose levels within a remarkably narrow range.
[0008] Among the islet cells are beta cells which produce and
secrete insulin. Insulin production and secretion by the beta cells
is controlled by blood glucose levels. Insulin release increases as
blood glucose levels increase. Insulin promotes the uptake of
glucose by target tissues and, thus, prevents hyperglycemia by
shuttling glucose into tissues for storage.
[0009] Beta cell dysfunction and the concomitant decrease in
insulin production can result in diabetes mellitus. In Type 1
diabetes, the beta cells are completely destroyed by the immune
system, resulting in an absence of insulin producing cells
(Physician's Guide to Insulin Dependent [Type I] Diabetes Mellitus:
Diagnosis and Treatment, American Diabetes Association, 1988). In
Type 2 diabetes, the beta cells become progressively less efficient
as the target tissues become resistant to the effects of insulin on
glucose uptake. Type 2 diabetes is a progressive disease and beta
cell function continues to deteriorate despite on-going treatment
with any presently available agent (UK Prospective Study Group,
1995). Thus, beta cells are absent in people with Type 1 diabetes
and are functionally impaired in people with Type 2 diabetes.
[0010] Beta cell dysfunction currently is treated in several
different ways. In the treatment of Type 1 diabetes or the late
stages of Type 2 diabetes, insulin replacement therapy is used.
Insulin therapy, although life-saving, does not restore
normoglycemia, even when continuous infusions or multiple
injections are used in complex regimes. For example, postprandial
levels of glucose continue to be excessively high in individuals on
insulin replacement therapy. Thus, insulin therapy must be
delivered by multiple daily injections or continuous infusion and
the effects must be carefully monitored to avoid hyperglycemia,
hypoglycemia, metabolic acidosis, and ketosis.
[0011] Replacement of beta cells can be achieved with pancreatic
transplants. (Scharp et al., 1991; Warnock et al., 1991). Such
transplants, however, require finding a matching donor, surgical
procedures for implanting the harvested tissue, and graft
acceptance. After transplantation in a person with Type 1 diabetes,
on-going immunosuppression therapy is required because cell surface
antigens on the beta cells are recognized and attacked by the same
processes that destroyed the beta cells originally.
Immunosuppressive drugs, such as cyclosporin A, however, have
numerous side-effects, including the increase in potential for
infection. Transplantation, therefore, can result in numerous
complications.
[0012] People with Type 2 diabetes are generally treated with drugs
that stimulate insulin production and secretion from the beta
cells. A major disadvantage of these drugs, however, is that
insulin production and secretion is promoted regardless of the
level of blood glucose. Thus, food intake must be balanced against
the promotion of insulin production and secretion to avoid
hypoglycemia or hyperglycemia.
[0013] In recent years several new agents have become available to
treat Type 2 diabetes. These include metformin, acarbose and
troglitazone (see Bressler and Johnson, 1997). However, the drop in
hemoglobin A1c obtained by these newer agents is less than adequate
(Ghazzi et al., 1997), suggesting that they will not improve the
long-term control of diabetes mellitus.
[0014] Most recently, glucagon-like peptide-1 (GLP-1), a hormone
normally secreted by neuroendocrine cells of the gut in response to
food, has been suggested as a new treatment for Type 2 diabetes
(Gutniak et al., 1992; Nauck et al., J. Clin. Invest., 1993). It
increases insulin release by the beta cells even in subjects with
long-standing Type 2 diabetes (Nauck et al., Diabetologia, 1993).
GLP-1 treatment has an advantage over insulin therapy because GLP-1
stimulates endogenous insulin secretion, which turns off when blood
glucose levels drop (Nauck et al., Diabetologia, 1993; Elahi et
al., 1994). when blood glucose levels are high. GLP-1 promotes
euglycemia by increasing insulin release and synthesis, inhibiting
glucagon release, and decreasing gastric emptying (Nauck et al.,.
Diabetologia, 1993; Elahi et al., 1994; Wills et al., 1996; Nathan
et al., 1992; De Ore et al., 1997). GLP-1 also induces an increase
in hexokinase messenger RNA levels (Wang et al., Endocrinology
1995; Wang et al., 1996). GLP-1 is known to have a potent
insulin-secreting effect on beta cells (Thorens and Waeber, 1993;
Orskov, 1992) and to increase insulin biosynthesis and proinsulin
gene expression when added to insulin-secreting cell lines for 24
hours (Drucker et al., 1987; Fehmann and Habener, 1992). In studies
using RIN 1046-38 cells, twenty-four hour treatment with GLP-1
increased glucose responsiveness even after the GLP-1 had been
removed for an hour and after several washings of the cells
(Montrose-Rafizadeh et al., 1994). Thus, GLP-1 is an insulin
releasing agent and an insulinotropic agent (i.e., an agent that
increases insulin synthesis) known to have a prolonged effect on
beta cells. GLP-1 is a product of posttranlational modification of
proglucagon. The sequences of GLP-1 and its active fragments GLP-1
(7-37) and GLP-1 (7-36) amide are known in the art (Fehmann et al.,
1995).
[0015] GLP-1 receptors have been shown to be present in the gut and
in the pancreatic islets (Id.). The receptors belong to a family of
G-protein-linked receptors that includes glucagon, secretin, and
vasoactive intestinal peptide receptors. After binding of GLP-1 to
its receptor there is a rise in cAMP in beta cells of the islets of
Langerhans (Widmann et al., 1996), indicating that the receptor is
coupled to the adenyl cyclase system by a stimulatory G-protein. In
peripheral tissues, such as liver, fat and skeletal muscle,
however, no increase in cAMP with GLP-1 is seen, suggesting that
GLP-1 acts through a different system on peripheral tissues
(Valverde and Villanueva-Penacarrillo, 1996).
[0016] Exendin-4 is a peptide produced in the salivary glands of
the Gila Monster lizard (Goke et al., 1993). The amino acid
sequence for Exendin-4 is known in the art (Felmann et al. 1995).
Although it is the product of a uniquely non-mammalian gene and
appears to be expressed only in the salivary gland (Chen and
Drucker, 1997), Exendin-4 shares a 52% amino acid sequence homology
with GLP-1 and in mammals interacts with the GLP-1 receptor (Goke
et al., 1993; Thorens et al., 1993). In vitro, Exendin-4 has been
shown to promote insulin secretion by insulin producing cells and,
given in equimolar quantities, is more potent than GLP-1 at causing
insulin release from insulin producing cells.
[0017] In vivo studies using GLP-1 have been limited to the use of
single or repeated bolus injections or short-term infusions of
GLP-1 and subsequent evaluation of the insulin secreting effects.
In one such study, infusions of GLP-1 for two hours were tested in
patients with Type 1 diabetes for the ability of GLP-1 to promote
glucose uptake in muscle and release of glucose from the liver
(Gutniak et al., 1992). Therapeutic uses of GLP-1 for increasing
the release of insulin have been considered for Type 2 diabetes,
but not for Type 1 diabetes, since Type 1 diabetes is marked by an
absence of beta cells, the known target cell for GLP-1.
Furthermore, GLP-1 has known limitations as a therapeutic agent in
the treatment of diabetes because it has a short biological
half-life (De Ore et al., 1997), even when given by a bolus
subcutaneously (Ritzel et al., 1995). Exendin-4 has not been used
previously in in vivo studies. Thus, studies to date have never
suggested that either GLP-1 or Exendin-4 is therapeutically
effective on pancreatic function in people with Type 1 diabetes or
that there are GLP-1 or Exendin-4 target cells in the pancreas
other than the beta cells.
SUMMARY OF THE INVENTION
[0018] It is an object of the present invention to overcome or
reduce the above stated problems with the prior art by providing a
population of insulin producing cells made by a process comprising
contacting non-insulin producing cells with a growth factor
selected from the group consisting of GLP-1 or Exendin-4, growth
factors having amino acid sequences substantially homologous to
GLP-1 or Exendin-4, and fragments thereof. In addition, a method of
differentiating non-insulin producing cells into insulin producing
cells, comprising contacting the non-insulin producing cells with a
growth factor selected from the group consisting of GLP-1 or
Exendin-4, growth factors having amino acid sequences substantially
homologous to GLP-1 or Exendin-4, and fragments thereof is
provided. Further provided is a method of enriching a population of
cells for insulin-producing cells, comprising contacting the
population of cells with a growth factor that promotes
differentiation of non-insulin producing cells into
insulin-producing cells.
[0019] Also provided is a method of treating diabetes in a subject
diagnosed with Type 1 diabetes, comprising administering to the
subject a growth factor selected from the group consisting of GLP-1
or Exendin-4, growth factors having amino acid sequences
substantially homologous to GLP-1 or Exendin-4, and fragments
thereof by continuous infusion for at least twenty-four hours. The
present invention further overcomes the prior art by providing a
method of treating diabetes in a subject, comprising obtaining
non-insulin producing cells from the subject being treated or from
a donor; contacting the non-insulin producing cells with a growth
factor, thereby promoting differentiation of non-insulin producing
cells into insulin-producing cells; and administering the
insulin-producing cells that were promoted to differentiate from
non-insulin producing cells to the diabetic subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 shows plasma insulin levels in 3 month and 22 month
old animals. GLP-1 (0.2 nmol/kg) was given intravenously to fasted,
anesthetized animals.
[0021] FIG. 2 shows plasma glucose levels during a glucose
tolerance in 22 month old animals. GLP-1-treated animals received
1.5 pmol/kg/min for 48 hrs by subcutaneous infusion. Controls were
infused with saline. Glucose (1 g/kg) was given ip and the blood
glucose measured at the times indicated. The results are a mean
(.+-.SEM) of 6 treated and 6 control animals. Repeated measures
analysis of variance from 0-30 min showed a value of p<0.05.
Asterisks indicate: * p<0.05, **p<0.01, as determined by
unpaired Student's t test.
[0022] FIG. 3 shows plasma insulin levels during a glucose
tolerance test in 22 month old animals. GLP-1-treated animals
received 1.5 pmol/kg/min for 48 hrs by subcutaneous injection.
Controls were infused with saline. Glucose (1 g/kg) was given ip
and serum insulin measured at the times indicated. The results are
mean (.+-.SEM) of 6 treated and 6 control animals. Repeated
measures analysis of variance from 0-30 min showed a value of
p<0.05. The asterisk indicates a p<0.01, as determined by
unpaired Student's t test.
[0023] FIG. 4 shows the fold increase in islet insulin content
after 48 hrs infusion of saline (control, 7 animals) or GLP-1 (1.5
pmol/kg/min, 7 animals) in 22 month old rats. **p<0.01 by
unpaired Student's t test.
[0024] FIG. 5 shows insulin mRNA levels in pancreata from control,
GLP-1-, Ex+GLP-1-, and Ex-treated 22 month old animals. Each sample
represents an individual pancreas, with four animals in each
treatment group.
[0025] FIG. 6 shows GLUT2 mRNA levels in pancreata from control,
GLP-1-, Ex+GLP-1-, and Ex-treated 22 month old animals. Each sample
represents an individual pancreas, with four animals in each
treatment group.
[0026] FIG. 7 shows glucokinase mRNA in pancreata from control,
GLP-1-, Ex+GLP-1-, and Ex-treated 22 month old animals. Each sample
represents an individual pancreas, with four animals in each
treatment group.
[0027] FIG. 8 shows plasma insulin concentrations in fasted
anesthetized rats after intravenous (iv) boli of GLP-1 (0.4
nmol/kg) and exendin-4 (0.4 nmol/kg) in 100 .mu.l NaCl. NaCl (100
.mu.l) was also given iv to a control group. Values are expressed
as mean.+-.SEM (n=6 per group).
[0028] FIG. 9 shows insulin concentrations in fasted anaesthetized
rats 2 min after intravenous boli of exendin-4 at the
concentrations shown. Values are expressed as mean.+-.SEM (n=6 per
exendin-4 concentration).
[0029] FIG. 10 shows the effects of GLP-1 (1 nM) and exendin-4 (1
nM) treatment for 1 hr on intracellular cAMP levels in islets of
Langerhans. Given are means.+-.SEM of 4 experiments, each done in
triplicate. Exendin-4 was more effective than GLP-1 (p<0.01).
Note that after washing some of the islets in buffer following the
hr in the presence of the peptides and then removing the islets
approximately 15 min later cAMP levels had returned to
baseline.
[0030] FIG. 11 shows a photograph of cages housing the diabetic
mice taken after nine weeks of treatment with exendin-4 (24
nmol/kg) or NaCl intraperitoneally daily, 24 hours after the
previous bedding change. Diabetic mice were housed two per cage.
Bedding was changed every 24 hours for the diabetic animals after
the first few weeks of treatment. The cage on the right contained
the exendin-4-treated animals while the cage on the left contained
the NaCl-treated animals.
[0031] FIG. 12 shows hemoglobin A1c levels in the diabetic and
non-diabetic mice given either exendin-4 (24 nmol/kg) or normal
saline intraperitoneally daily for 12-13 weeks. Values are
expressed as mean.+-.SEM (n=9-10 per group).
[0032] FIG. 13 shows fasting glucose and insulin concentrations in
the diabetic and non-diabetic mice given either exendin-4 (24
mmol/kg) or normal saline intraperitoneally daily for 12-13 weeks.
Values are expressed as mean.+-.SEM (n=9-10 per group).
[0033] FIG. 14 shows CCK concentration-response curve of amylase
release from AR42J cells. Cells were treated with CCK at the
concentrations shown for 50 min. Amylase values are expressed as a
percentage of the released amylase into the medium over the total
amylase activity of the cells. Results are the mean.+-.SEM of 15
experiments.
[0034] FIG. 15 shows the effects of glucagon (10 nM), GLP-1 (10
nM), and insulin (100 nM).+-.CCK (1 nM), on amylase release from
AR42J cells. Dexamethasone-induced AR42J cells were incubated for
50 min in presence of the hormones. Amylase values are expressed as
a percentage of the released amylase into the medium over the total
amylase activity of the cells. Results are mean.+-.SEM of 20
experiments, * p<0.05, **p<0.01, treatment vs. no treatment.
a=p<0.01.
[0035] FIG. 16 shows the effect of 50 min of treatment with
8-Bromo-cAMP (100 nM) on amylase release from AR42J cells. Amylase
values are expressed as a percentage of the released amylase into
the medium over the total amylase activity of the cells. Results
are mean.+-.SEM of 3 experiments, * p<0.05, treatment vs. no
treatment.
[0036] FIG. 17 shows the effects of ryanodine (RY) and thapsigargin
(TG) in the presence or absence of CCK on amylase release from
AR42J cells. Amylase values are expressed as a percentage of the
released amylase into the medium over the total amylase activity of
the cells. RY and TG were added 30 min prior to addition of CCK,
which was then added for 50 min. Results are mean.+-.SEM of 3
experiments, **p<0.01.
[0037] FIG. 18 shows the time course of the actions of vanadate (1
mM) (.DELTA.) and genestein (300 .mu.M) (.circle-solid.) on CCK (1
nM)-mediated amylase release from AR42J cells. Amylase release from
CCK-treated (1 nM) (.smallcircle.) cells or control (no treatment)
(.quadrature.) cells is also shown. Amylase values are expressed as
a percentage of the released amylase into the medium over the total
amylase activity of the cells. Results are the mean.+-.SEM of 4
experiments. * p<0.05, **p<0.01, vanadate or genestein plus
CCK treatment vs. CCK treatment alone.
[0038] FIG. 19 shows the effects of CCK on intracellular free
[Ca.sup.2+].sub.i in single AR42J cells. The bar indicates the time
of exposure to 10 nM CCK in three different cells. FIG. 19A shows
the typical [Ca.sup.2+].sub.i response observed in at least 85% of
the cells. FIG. 19B shows that the [Ca.sup.2+].sub.i response to
CCK is almost completely abolished following 60 min exposure to 10
.mu.M ryanodine (RY) and 500 nM thapsigargin (TG). FIG. 19C shows
that the [Ca.sup.2+].sub.i transient is abbreviated by reduction of
extracellular Ca.sup.2+ during exposure to CCK.
[0039] FIG. 20 shows the effects of GLP-1 on [Ca.sup.2+].sub.i and
CCK-induced [Ca.sup.2+].sub.i transients in single AR42J cells. The
same cell was studied in A-C. FIG. 20A shows that exposure to 1 nM
GLP-1 induced small, slow, prolonged [Ca.sup.2+].sub.i transients
in approximately 50% of AR42J cells. The reduction in the amplitude
of the subsequent exposure to 10 .mu.M CCK is shown in FIG. 20B.
FIG. 20C shows that the amplitude of the [Ca.sup.2+].sub.i
transient is further reduced in response to a second exposure to
CCK applied in <10 min.
[0040] FIG. 21 shows the effects of glucagon and 8-bromo-cAMP
(8BcAMP) on [Ca.sup.2+].sub.i in single AR42J cells. FIG. 21A shows
that glucagon (10 nM) induced slow, small, prolonged
[Ca.sup.2+].sub.i transients in approximately 70% of cells. FIG.
21B shows that in cells treated with 10 nM glucagon for 3-10 min,
the subsequent [Ca.sup.2+].sub.i transients induced by 10 nM CCK
showed a slow rate of rise as well as a prolonged relaxation phase.
FIG. 21C shows that brief (1-5 min) exposures to 100 nM 8BcAMP
attenuated the relaxation of CCK-induced [Ca.sup.2+].sub.i
transients.
[0041] FIG. 22 shows the effects of GLP-1 (10 nM), glucagon (10 nM)
and CCK (1 nM) treatments.+-.IBMX (100 nM) for 50 min on
intracellular cAMP levels in AR42J cells. Results are mean.+-.SEM
of 3 experiments, *p<0.05.
[0042] FIG. 23 shows RT-PCR of GLP-1 receptors in AR42J cells and
rat pancreas. cDNA was amplified for 30 cycles using primers in the
5'- and 3'-end of the rat pancreatic GLP-1 receptor. PCR products
were resolved on a 1% agarose gel and visualized using ethidium
bromide. From left to right; Lane 1, DNA marker; Lane 2, blank;
Lane 3, AR42J cells; Lane 4, rat pancreas; Lane 5, water control.
In Lanes 3 and 4 we see the expected 928 bp band, corresponding to
the GLP-1 receptor.
[0043] FIG. 24 shows western blot analysis of GLP-1 receptor
expression in AR42J (Lane 1, 2) and RIN 1046-38 (Lane 3, 4) cells.
Cells were solubilized and GLP-1 receptors were detected after
immunoprecipitation and Western blotting with antibody to the
amino-terminus of the GLP-1 receptor. The positions of the
molecular markers, in kDa, are on the right. The 65 and 46 kDa
bands have been shown to correspond to the mature and
core-glycosylated GLP-1 receptors, respectively (28).
[0044] FIG. 25 shows protein tyrosine phosphorylation in AR42J
cells in response to various stimuli. A representative
anti-phosphotyrosine immunoblot of total cellular proteins from
un-treated (control) cells and 5 min-treated cells as indicated
(n=3). Note the increase in tyrosine phosphorylation with CCK and
sodium fluoride (NaF) of 46, 66, 120 and 190 kDa bands. GLP-1 did
not have any effect on those proteins. Insulin caused increased
phosphorylation of 97 kDa band, corresponding to the insulin
receptor .beta.-subunit.
[0045] FIG. 26 shows immunocytochemistry of AR42J cells. AR42J
cells were fixed with glutaraldehyde, and incubated with
anti-insulin or anti-glucagon antibody from Linco at a dilution of
1:300. FIG. 26A shows control AR42J cells, anti-insulin antibody.
FIG. 26B shows GLP-1 (10 nM)-treated cells for 48 hours,
anti-insulin antibody. FIG. 26C shows GLP-1 (10 nM)-treated AR42J
cells for 72 hours, anti-insulin antibody. FIG. 26D shows RIN
1046-38 insulinoma cells, anti-insulin antibody. FIG. 26E shows
control cells, anti-glucagon antibody. FIG. 26F shows GLP-1 (10
nM)-treated cells for 48 hours, anti-glucagon antibody.
[0046] FIG. 27 shows the effect of time on the induction of
glucagon and insulin by GLP-1 (10 nM) production in AR42J cells.
For this experiment, cells were plated on the coverslips as
described herein, all on the same day. They were then stained with
anti-insulin or anti-glucagon antibody on the days indicated. This
has now been repeated numerous times (at least 5 times) on
different days and insulin and glucagon have always been
present.
[0047] FIG. 28 shows expression of mRNAs for insulin and glucagon
using RT-PCR. FIG. 28A shows insulin mRNA at 187 bp. FIG. 28B shows
glucagon mRNA at 236 bp. GLP-1 (1 nM) treatment was for 3 days.
[0048] FIG. 29 shows the effect of GLP-1 and exendin-4 in the
presence or absence of a protein kinase C inhibitor in AR42J cells
from one representative experiment, which was repeated 3 times.
FIG. 29A is the autoradiogram and FIG. 29B represents the
densitometry readings (relative units). Cells were plated at a
density of 10.sup.5/well in 60 mm dishes, lysed and clarified
lysates were then immunoprecipitated with anti-ERK antibody. The
immune pellets were analyzed for ERK activity as described in
herein. Lane 1, Control AR42J cells. Lane 2, GLP-1 (10 nM)-treated
AR42J cells for 3 days. Lane 3, exendin-4 (0.1 nM)-treated cells
for 3 days. Lane 4, GLP-1 (10 nM)-plus exendin-4 (0.1 nM)-treated
cells for 3 days. Lane 5, GLP-1 (10 nM)-plus exendin-4-plus PKI
(300 .mu.M)-treated cells for 3 days. Lane 6, exendin-4 (0.1)-plus
PKI (300 .mu.M)-treated cells for 3 days. Lane 7, GLP-1 (10
nM)-plus PKI (300 .mu.M)-treated cells for 3 days. Note that
exendin-4 (0.1 nM) is approximately equivalent to GLP-1 (10
nM).
[0049] FIG. 30 shows the dose-response effect of GLP-1 on amylase
release from dexamethasone-treated AR42J cell. After 3 days
treatment with different concentration of GLP-1 the AR42J cells
were washed and 1 nM CCK was added. The cells were incubated for
another 50 min and the samples were collected for amylase assay.
N=4, Mean.+-.SEM.
DETAILED DESCRIPTION OF THE INVENTION
[0050] As used in the claims, "a" can mean one or more.
[0051] The present invention provides a population of insulin
producing cells made by a process comprising contacting non-insulin
producing cells with a growth factor selected from the group
consisting of GLP-1 or Exendin-4, growth factors having amino acid
sequences substantially homologous thereto, and fragments thereof.
Non-insulin producing cells, including primary acinar cells, acinar
cell lines (e.g., AR42J), and stem cells, that were not previously
thought to have GLP-1 receptors and not previously thought to be
capable of producing insulin can respond to GLP-1 and Exendin-4,
growth factors having amino acid sequences substantially homologous
thereto, and fragments thereof by differentiating into insulin
producing cells. The effect is to increase the number of
insulin-producing cells, an effect that is desirable in the
treatment of diabetes mellitus.
[0052] As used herein, "insulin producing cells" includes cells
that synthesize (i.e., transcribe the insulin gene, translate the
proinsulin mRNA, and modify the proinsulin mRNA into the insulin
protein), express (i.e., manifest the phenotypic trait carried by
the insulin gene), or secrete (release insulin into the
extracellular space) insulin in a constitutive or inducible manner.
Examples of known insulin producing cells include beta cells, which
are located in the pancreatic islets in vivo. In order to secrete
insulin, an insulin producing cell also must express IDX-1.
[0053] A population of insulin producing cells made by the present
invention may contain cells (e.g., beta cells) that produce insulin
without the use of the present methods and other cell types. The
novelty of the present composition and methods is not negated by
the presence of cells in the population that produce insulin
naturally (e.g., beta cells). It is also contemplated that the
population of insulin producing cells may also contain non-insulin
producing cells.
[0054] By "non-insulin producing cells" is meant any cell that does
not naturally synthesize, express, or secrete insulin
constitutively or inducibly. Thus, the term "non-insulin producing
cells" as used herein excludes beta cells. Examples of non-insulin
producing cells that can be used in the methods of the present
invention include pancreatic non-beta cells, such as amylase
producing cells, acinar cells, cells of ductal adenocarcinoma cell
lines (e.g., CD18, CD11, and Capan-1 cells (see Busik et al., 1997;
Schaffert et al. 1997), and stem cells. Non-pancreatic cells could
also be used, for example, non-pancreatic stem cells and cells of
other endocrine or exocrine organs, including, for example,
pituitary cells. The non-insulin producing cells can be mammalian
cells or, even more specifically, human cells. Examples of the
present method using mammalian pancreatic non-islet, pancreatic
amylase producing cells, pancreatic acinar cells, and stem cells
are provided herein. Stem cells can include pancreatic stem cells
and non-pancreatic stem cells that have been promoted to produce
IDX-1, Beta 2/NeuroD, and E47. Pancreatic stem cells include duct
epithelial precursor cells which give rise to both islet and acinar
cells.
[0055] The non-insulin producing cells must have GLP-1 receptors or
receptors substantially similar to GLP-1 receptors in order to
differentiate into insulin producing cells. Preferably, the
non-insulin producing cell could also show, upon contact with the
growth factor, an increase in intracellular calcium and ERK/MAPK
activity and activation of PKC.
[0056] As used in the present application, "growth factor" means a
substance that can differentiate a non-insulin producing cell into
an insulin producing cell. Preferably the growth factor will be one
of a group of insulinotropic growth factors, including, for
example, GLP-1, exendin-4, betacellulin, Hepatocyte Scatter Factor
(HSF) and activin-A, or combinations thereof, excluding
betacellulin and activin-A used together and excluding HSF and
Activin-A used together. Preferably, greater than 10% of the
non-insulin producing cells will differentiate into insulin
producing cells upon contact with the growth factor; and, more
preferably, at least about 20%, 30%, 40%, 50%, or more will
differentiate into insulin producing cells upon contact with the
growth factor. Thus, a population of insulin producing cells made
in vitro according to the present method can comprise as low as 11%
and up to 100% insulin producing cells.
[0057] By "amino acid sequences substantially homologous" to GLP-1
or exendin-4 is meant polypeptides that include one or more
additional amino acids, deletions of amino acids, or substitutions
in the amino acid sequence of GLP-1 or exendin-4 without
appreciable loss of functional activity as compared to GLP-1 or
exendin-4 in terms of the ability to differentiate insulin
producing cells from non-insulin producing cells. For example, the
deletion can consist of amino acids that are not essential to the
presently defined differentiating activity and the substitution(s)
can be conservative (i.e., basic, hydrophilic, or hydrophobic amino
acids substituted for the same). Thus, it is understood that, where
desired, modifications and changes may be made in the amino acid
sequence of GLP-1 and Exendin-4, and a protein having like
characteristics still obtained. It is thus contemplated that
various changes may be made in the amino acid sequence of the GLP-1
or Exendin-4 amino acid sequence (or underlying nucleic acid
sequence) without appreciable loss of biological utility or
activity and possibly with an increase in such utility or
activity.
[0058] The term "fragments," as used herein regarding GLP-1,
Exendin-4, or growth factors having amino acid sequences
substantially homologous thereto means a polypeptide sequence of at
least 5 contiguous amino acids of either GLP-1, Exendin 4, or
growth factors having amino acid sequences substantially homologous
thereto, wherein the polypeptide sequence has the differentiating
function of GLP-1 and Exendin-4 as described herein. The present
fragment may have additional functions that can include
antigenicity, binding to GLP-1 receptors, DNA binding (as in
transcription factors), RNA binding (as in regulating RNA stability
or degradation). Active fragments of GLP-1 can include, for
example, GLP-1 (7-36) amide (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR (SEQ ID
NO:1)); GLP-1 (7-37) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG (SEQ ID
NO:2)); GLP-1 (7-35) (HAEGTFTSDVSSYLEGQAAKEFIAWLVKG (SEQ ID NO:
3)); GLP-1 (7-34) (HAEGTFTSDVSSYLEGQAAKEFIAWLVK (SEQ ID NO:4));
GLP-1 (7-33) (HAEGTFTSDVSSYLEGQAAKEFIAWLV (SEQ ID NO:5)); GLP-1
(7-32) (HAEGTFTSDVSSYLEGQAAKEFIAWL SEQ ID NO:6)); GLP-1 (7-31)
(HAEGTFTSDVSSYLEGQAAKEFIAW SEQ ID NO:7)); and GLP-1 (7-30)
(HAEGTFTSDVSSYLEGQAAKEFIA SEQ ID NO:8)). Active fragments of
Exendin-4 can include, for example, Exendin-4 (1-39)
(HGEGTFTSDLSKQMEEEAVRLFEWLKNGGPSSGAPPPS (SEQ ID NO:9)); Exendin-4
(1-38) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPP (SEQ ID NO: 10));
Exendin-4 (1-37) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPP (SEQ ID NO:
11)); Exendin-4 (1-36) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAP (SEQ
ID NO: 12)); Exendin-4 (1-35) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGA
(SEQ ID NO: 13)); Exendin-4 (1-34)
(HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSG (SEQ ID NO:14)); Exendin-4
(1-33) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSS (SEQ ID NO: 15));
Exendin-4 (1-32) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPS (SEQ ID NO:
16)); Exendin-4 (1-31) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGP (SEQ ID
NO:17)); and Exendin-4 (1-30) (HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG (SEQ
ID NO:18)).
[0059] Other fragments and modified sequences of GLP-1 are known in
the art (U.S. Pat. No. 5,614,492; U.S. Pat. No. 5,545,618; European
Patent Application, Publication No. EP 0658568 A1; WO 93/25579).
Similar fragments and modified sequences of exendin-4 can be easily
extrapolated. It is expected that the following residues in GLP-1
(residue number in superscript) and exendin-4 (residue number in
parentheses and superscript) should be included in a fragment since
these residues are highly conserved and are important for receptor
binding: H.sup.7(1), G.sup.10(4), F.sup.12(6), T.sup.13(7),
D.sup.15(9). Thus, additional fragments or modified sequences can
be easily made that exclude or alter amino acids of GLP-1 and
exendin-4, other than these 5. Because the differentiation activity
disclosed herein is easy to assess, the determination that a
fragment is within the scope of the invention is routine.
[0060] The present invention provides a population of insulin
secreting cells made by the methods described herein. Because IDX-1
expression is required for insulin secretion from a cell, the
non-insulin producing cells that can be used to make insulin
secreting cells should include cells that express IDX-1
constitutively or upon stimulation with a growth factor or by
transfecting the cell with a nucleic acid encoding IDX-1 prior to,
during, or after treatment of the non-insulin producing cells with
the growth factor.
[0061] The present invention also provides a growth factor that can
differentiate insulin producing cells from non-insulin producing
cells. Such growth factors include but are not limited to GLP-1,
Exendin-4, or growth factors having amino acid sequences
substantially homologous thereto, and fragments thereof.
Differentiation could occur in vivo or in vitro upon contact of the
non-insulin producing cell with the growth factor. The contact
could be one time by bolus, one time by continuous infusion, or
repeatedly by bolus or continuous infusion.
[0062] The present invention also provides a method of screening
for growth factors that differentiate an insulin producing cell
from a non-insulin producing cell. More specifically, the screening
method involves the steps of (1) contacting the growth factor to be
screened with a non-insulin producing cell, (2) evaluating the
non-insulin producing cell for characteristics of insulin producing
cells, and (3) identifying the growth factors that differentiate
insulin producing cells from non-insulin producing cells. The
preferred characteristics of insulin producing cells include the
ability to transcribe the insulin gene, the ability to translate
the insulin mRNA, the ability to release or secrete insulin, the
ability to store insulin, the ability to sense levels of glucose,
and the ability to release insulin in a regulated fashion. Since
expression of the transcription factors IDX-1, Beta 2/NeuroD, and
E47 are believed to be necessary for production of insulin, these
factors will also typically be expressed in the insulin-producing
cell of the invention.
[0063] By "contacting" is meant an instance of exposure of the
extracellular surface of a cell to a substance at physiologically
effective levels. A cell can be contacted by a growth factor, for
example, by adding the growth factor to the culture medium (by
continuous infusion, by bolus delivery, or by changing the medium
to a medium that contains growth factor) or by adding the growth
factor to the intracellular fluid in vivo (by local delivery,
systemic delivery, intravenous injection, bolus delivery, or
continuous infusion). The duration of "contact" with a cell or
group of cells is determined by the time the substance, in this
case a growth factor, is present at physiologically effective
levels in the medium or extracellular fluid bathing the cell. GLP-1
has a short half-life of several minutes, whereas Exendin-4's
half-life is substantially longer, on the order of hours. A bolus
of GLP-1 would, therefore, have contact with the cell for minutes,
and a bolus of Exendin-4 would contact the cell for hours.
[0064] The contacting step in the methods of the present invention
can take place in vitro. For example, in a transplantation
protocol, ex vivo methods can be employed such that non-insulin
producing cells are removed from a donor (e.g., the subject being
treated) and maintained outside the body according to standard
protocols well known in the art (see Gromada et al., 1998). While
maintained outside the body, the cells could be contacted with the
growth factor and the cells subsequently infused (e.g., in an
acceptable carrier) or transplanted using methods well known in the
art into the donor subject or a subject different from the donor
subject.
[0065] Alternatively, the contacting step of the present invention
can take place in vivo. Methods for administering GLP-1, Exendin-4
or related growth factors are provided herein. The GLP-1,
Exendin-4, or related growth factors are administered systemically,
including, for example, by a pump, by an intravenous line, or by
bolus injection (Gutniak et al., 1992; European Patent Application,
Publication No. 0619322 A2; U.S. Pat. No. 5,614,492; U.S. Pat. No.
5,545,618). Bolus injection can include subcutaneous,
intramuscular, or intraperitoneal routes.
[0066] Non-insulin producing cells begin to differentiate into
insulin producing cells after about twenty-four hours of contact
with GLP-1 or Exendin-4, growth factors having amino acid sequences
substantially homologous thereto, or fragments thereof. The maximum
number of cells that differentiate into insulin-producing cells
usually have done so after about seven days of contact.
Interestingly, the new insulin producing cells continue to show the
capacity to produce insulin even after contact with GLP-1 or
Exendin-4, their fragments or related growth factors is
discontinued. The new insulin producing cells show the capacity to
produce insulin at least up to 2 weeks after contact is
discontinued.
[0067] Thus, the contacting step will typically be for at least
twenty-four hours. By "at least twenty-four hours," is meant
twenty-four hours or greater. Specifically, the non-insulin
producing cells can be contacted with the growth factor for 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60 hours up to 3, 4, 5, 6, 7, or more days or any particular
intervening time in hours or minutes within the above range.
Preferably the non-insulin producing cells will be contacted with
the growth factor for seven days.
[0068] The dosages of GLP-1, Exendin-4, their active fragments or
related growth factors to be used in the in vivo or in vitro
methods and processes of the invention preferably range from about
1 pmoles/kg/minute to about 100 nmoles/kg/minute for continuous
administration and from about 1 nmoles/kg to about 40 nmoles/kg for
bolus injection. Preferably, the dosage of GLP-1 in in vitro
methods will be 10 pmoles/kg/min to about 100 nmoles/kg/min, and in
in vivo methods from about 0.003 nmoles/kg/min to about 48
nmoles/kg/min. More preferably, the dosage of GLP-1 in in vitro
methods ranges from about 100 picomoles/kg/minute to about 10
nanomoles/kg/minute, and in in vivo methods from about 0.03
nanomoles/kg/minute to about 4.8 nanomoles/kg/minute. The preferred
dosage of exendin-4 in in vitro methods is 1 pmoles/kg/min to about
10 nmoles/kg/mine, and in in vivo from about 1 pmole/kg to about
400 pmoles/kg for a bolus injection. The more preferred dosage of
exendin-4 in in vitro methods ranges from about 10 pmole/kg/minute
to about 1 mole/kg/minute, and in in vivo from about 10 pmoles/kg
to about 40 pmoles/kg for a bolus injection.
[0069] A method of differentiating non-insulin producing cells into
insulin producing cells, comprising contacting the non-insulin
producing cells with a growth factor selected from the group
consisting of GLP-1 or Exendin-4, growth factors having amino acid
sequences substantially homologous thereto, and fragments thereof
is provided. By "differentiating non-insulin producing cells into
insulin producing cells" is meant a change in the phenotypic
characteristics of the non-insulin producing cells such that the
affected cells have at least the phenotypic characteristic of
producing insulin. The affected cell may have all of the phenotypic
characteristics of a beta cell or may have less than all of the
phenotypic characteristics of a beta cell. The affected cell may
produce insulin but otherwise maintain the phenotypic
characteristics of the non-insulin producing cell. For example, a
non-insulin producing cell, such as a pancreatic amylase producing
cell (i.e., pancreatic acinar cell), that is contacted with GLP-1
or Exendin-4 can continue to express amylase, typical of an amylase
producing cell, but, unlike the typical amylase producing cell,
also produces insulin. Thus, a continuum between complete
phenotypic change and a single phenotypic change is possible. The
examples show the surprising result that insulin producing
capability can be conferred upon mature non-insulin secreting cells
(e.g., acinar cells). An increase in proliferation of non-insulin
producing cells may precede the differentiation of non-insulin
producing cells into insulin producing cells, and "differentiating"
is not meant to exclude any proliferation that accompanies the
change of the cell to an insulin producing phenotype.
[0070] Because of the importance of IDX-1, Beta 2/NeuroD, and E27
in the secretion of insulin, the present invention also provides a
method of differentiating non-insulin producing cells into insulin
producing cells that includes the additional step of transfecting
the non-insulin producing cell with a nucleic acid or nucleic acids
encoding IDX-1, Beta 2/NeuroD, and/or E27 prior to contacting the
non-insulin producing cell with GLP-1, Exendin-4, or similar growth
factor. Alternatively, an additional step can comprise transfecting
a cell already contacted with GLP-1 or Exendin-4, or a similar
growth factor with a nucleic acid or nucleic acids encoding IDX-1,
Beta 2/NeuroD, and/or E27. If the contacted cell is in vivo,
transfection could be achieved by retrograde perfusion of plasmid
DNA for IDX-1 into the secretory duct of the pancreas (see Goldfine
et al., 1997). Additionally, in some cases, expression or IDX-1,
Beta 2/NeuroD, and E47 can result from the application of certain
proteins to non-IDX expressing cells, including, for example, stem
cells.
[0071] The present invention provides a method of enriching a
population of cells for insulin-producing cells, comprising
contacting the population of cells with a growth factor that
promotes differentiation of non-insulin producing cells into
insulin-producing cells. The population of cells produced by this
process is expanded in the number of insulin producing cells and
can be used in the treatment methods described herein.
[0072] The present invention further provides a method of promoting
pancreatic amylase producing cells to produce both insulin and
amylase, comprising contacting the pancreatic amylase producing
cells with a growth factor selected from the group consisting of
GLP-1 or Exendin-4, growth factors having amino acid sequences
substantially homologous thereto, and fragments thereof. An example
of this method is provided in the examples.
[0073] The present invention further provides a method of treating
diabetes in a subject diagnosed with Type 1 diabetes, comprising
administering to the subject a growth factor selected from the
group consisting of GLP-1, growth factors having amino acid
sequences substantially homologous thereto, and fragments thereof
by continuous infusion for at least twenty-four hours.
Alternatively, the growth factor can be selected from the group
consisting of Exendin-4, growth factors having amino acid sequences
substantially homologous thereto, and fragments thereof. Since
Exendin-4 has a fairly long half-life, compared to GLP-1, it can be
administered by bolus at least once. The treatment methods are
effective to treat diabetes in a subject with Type 1 diabetes,
because the growth factor promotes the differentiation of
non-insulin producing cells in the subject into insulin producing
cells, as described in detail herein.
[0074] The subject of the invention can include individual humans,
domesticated animals, livestock (e.g., cattle, horses, pigs, etc.),
pets (like cats and dogs).
[0075] By "diabetes" is meant diabetes mellitus, a metabolic
disease characterized by a deficiency or absence of insulin
secretion by the pancreas. As used throughout, "diabetes" includes
Type 1, Type 2, Type 3, and Type 4 diabetes mellitus unless
otherwise specified herein.
[0076] The present invention also provides a method of treating
diabetes in a subject, comprising obtaining non-insulin producing
cells from the subject being treated or from a donor; contacting
the non-insulin producing cells with a growth factor in vitro,
thereby promoting differentiation of non-insulin producing cells
into insulin-producing cells; and administering the
insulin-producing cells that were promoted to differentiate from
non-insulin producing cells to the diabetic subject. In the method
of treating diabetes, wherein the non-insulin producing cells are
from a donor, the donor can be a cadaver. As a further embodiment
of the present invention, the non-insulin producing cells can be
allowed to proliferate in vitro prior to contact with the growth
factor. Preferably, promoting differentiation of non-insulin
producing cells into insulin-producing cells will result in greater
than about 20% differentiation of non-insulin producing cells into
insulin-producing cells. Even more preferably, greater than about
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or
90% of the treated cells will differentiate into insulin-producing
cells.
[0077] Altering the surface antigens of the insulin producing cells
obtained by the differentiation of the non-insulin producing cells
into the insulin producing cells, can reduce the likelihood that
the insulin producing cells will cause an immune response. The
cells with altered surface antigens can then be administered to the
diabetic subject. The cell surface antigens can be altered prior
to, during, or after the non-insulin producing cells are
differentiated into insulin-producing cells.
[0078] The present invention also provides a method of
differentiating endothelial cells into smooth muscle cells,
comprising contacting the endothelial cells with a growth factor
selected from the group consisting of GLP-1 or Exendin-4, growth
factors having amino acid sequences substantially homologous
thereto, and fragments thereof.
[0079] The present invention will now be illustrated with reference
to the following examples.
EXAMPLES OF THE INVENTION
Example 1
[0080] As GLP-1 in cultured insulinoma cells is known to impact
positively on insulin secretion, insulin synthesis and insulin
messenger RNA, GLP-1's effects on aging Wistar rats were
evaluated.
[0081] Materials. GLP-1 and exendin [9-39] (Ex), a peptide receptor
antagonist of GLP-1, were purchased from Bachem (King of Prussia,
Pa.). Chemical reagents were from Sigma (St Louis, Mo.), unless
otherwise stated.
[0082] Animals. Three month (young) and 22 month (old) old Wistar
rats from the Wistar colony in the NIA (Baltimore, Md.) were used.
They had been maintained on rat chow and fed ad libitum. All our
aged rats are the offspring of ten founder families maintained at
the NIA.
[0083] Protocols. To insure that old animals were capable of
responding to GLP-1, we carried out an acute experiment with an
intravenous bolus of GLP-1. Six old (22 months) and 6 young (3
months) Wistar rats were fasted overnight. Following anesthesia
with 50 mg/kg pentobarbital, a catheter was placed in the femoral
artery for blood sampling, and a bolus of GLP-1 (0.2 mmol/kg) was
given into the saphenous vein over 30 sec. Blood (taken at 2, 4, 7,
and 10 min) was collected for insulin determination.
[0084] The rats were implanted with an Alzet micro-osmotic pump
(Alza Corp., Palo Alto, Calif.) in the interscapular region for 48
hrs. In the treated group GLP-1 was delivered at the rate of 1.5
and Ex at 15 pmol/kg.sup.-1.min.sup.-1. It has been shown that in
order to prevent GLP-1's insulinotropic effect a 10-fold higher
concentration of Ex is required (Wang et al., J. Clin. Invest.,
1995). Control animals received normal saline in their pumps and
received their infusion for the same length of time.
[0085] For glucose tolerance testing, after the pumps were in place
36 hrs (n=6 each for control and GLP-1 treatment), the rats were
fasted overnight, anesthetized with 50 mg/kg pentobarbital, and the
pumps were removed, giving a total GLP-1 infusion time of 48 hrs. A
catheter was placed in the femoral artery for blood sampling and
blood was collected for GLP-1 measurement. An intraperitoneal (ip)
glucose tolerance test (IPGTT, Ig/Kg BW) was administered 120 min
after removal of the pumps. Blood samples were obtained at 15, 30,
45, 60, and 90 min in order to estimate glucose and insulin levels.
Blood (200 .mu.l) was drawn into heparinized tubes containing EDTA
for glucose and insulin determination.
[0086] For intraislet content of insulin, 14 rats were used. Seven
were treated with GLP-1, and seven with saline, as described above.
After 48 hrs, preceded by an overnight fast, the animals were
sacrificed and islets of Langerhans harvested as described
previously (Perfetti et al., 1995; Egan et al., 1991). We then
measured the intraislet insulin content in 50 islets picked at
random from each individual pancreas. Picked islets were
centrifuged, any residual medium was removed, the pellet was
suspended in ice-cold acid-ethanol (500 ul) and homogenized. After
centrifugation of the homogenate (1400.times.g, 4.degree. C.) the
supernatant was collected for measurement of intracellular insulin.
The pellet was dissolved in formic acid and protein content was
determined.
[0087] Assays. Plasma glucose was measured by the glucose oxidase
method (Egan et al., 1991). Insulin and GLP-1 were measured by RIA
as previously published (Wang et al., Endocrinology, 1995; Nathan
et al., 1992). The amount of cellular proteins was measured using
the Bradford method (Bio-Rad Richmond, Calif.) using bovine
.gamma.-globulin as standard.
[0088] RNA isolation and quantitation of endocrine pancreatic
mRNAs. Whole pancreata of rats that had been subjected to 48 hrs
infusion with GLP-1 or saline were used to extract total RNA. After
an overnight fast, animals were sacrificed, pancreata were removed
and frozen in liquid nitrogen as quickly as possible. RNA was
extracted by homogenization in guanidinium isothiocyanate, followed
by ultracentrifugation on a 5.7 M cesium chloride cushion (Glisin
et al., 1974; Chigwin et al., 1979). Poly-A RNA was then prepared
from total RNA by affinity chromatography using oligo (dT) columns
(Biolabs INC, Beverly, Mass.). RNA was quantified by
spectrophotometric analysis at 260 nM. Slot-blot analysis using
poly-A RNA was used for quantitation of mRNA levels of glucokinase,
which is the main glucose sensor of the beta cell (Matschinsky,
1990), the three hexokinases, GLUT2, the major glucose transporter
of the beta cell (Mueckler, 1990), and insulin. Four micrograms of
poly-A RNA were diluted in 50 .mu.l TE buffer (Tris-HCL 10 mM, EDTA
1 mM, pH 7.4), 20 .mu.l of 37% formaldehyde and 20 .mu.l of
10.times.SSC. Samples were incubated at 60.degree. C. for 15 min
and then diluted with 1 ml of ice-cold 10.times.SSC
(1.times.SCC=0.15 M NaCl+0.015 sodium citrate). Each well of
slot-blot minifolder was rinsed once with ice-cold 10.times.SSC and
300 .mu.l of sample per well was then loaded in triplicate onto the
membrane. Vacuum was applied to drain the samples through the
membrane, followed by three washes of the wells with ice-cold
10.times.SSC. Finally, membranes were baked for 2 hrs at 80.degree.
C. in a vacuum oven for RNA crosslinking.
[0089] The hybridizations with cDNA probes (rat insulin II from Dr.
S. J. Giddings, Washington University, St. Louis, Mo.; rat
glucokinase from Dr. M. A. Magnuson, Vanderbilt University,
Nashville, Tenn.; rat GLUT2 from Dr. M. J. Birnbaum, Harvard
Medical School, Boston, Mass.; and hexokinase 1, II, III cDNAs from
Dr. J. E. Wilson, Michigan State University, East Lancing, Mich.)
were carried out as previously described (Wang et al.,
Endocrinology, 1995; Wang et al., Mol. Cell. Endocrinol., 1996).
All cDNA probes were labeled with [.sup.32P] dCTP (Amersham Life
Science, Arlington Heights, Ill.) by random priming procedure using
Sequenase United States Biochemical, Cleveland, Ohio). An
oligonucleotide homologous to the poly-A tail of mRNAs was
synthesized on an Applied Biosystem DNA synthesizer
(5'GATGGATCCTGCAGAAGCTTTTTTTTTTTTTTTTTTTT3') and used to quantify
total cellular mRNA. Hybridization with oligo dT.sub.20 was carried
out in order to verify that an approximately equal amount of RNA
was used for each sample. Oligonucleotide probes were end-labeled
with [.sup.32P] .gamma.ATP (Amersham) using T4 polynucleotide
kinase (New England Biolabs, Beverly, Mass.). The hybridizations
with oligonucleotide probes were carried out as described before
(Wang et al., Endocrinology, 1995; Wang et al., Mol. Cell.
Endocrinol., 1996) and quantified using a Betascope 603 blot
analyzer (Betagen, Walthman, Mass.).
[0090] RNA isolation and quantitation of mRNAs in islets of
Langerhans. To confirm the changes seen in whole pancreata RNA was
isolated from islets of Langerhans from animals treated as
described above with GLP-1. Islets were isolated and RNA extracted
using the micromethod, previously described (Perfetti et al.,
1995). Approximately 5 .mu.g of total islet RNA was from one
pancreas. Slot-Blot analysis was carried out to quantitate mRNA
levels of the hexokinases, GLUT2, and insulin.
[0091] Statistical analysis The data were expressed as the
mean.+-.SEM. Significance of the insulin and glucose data obtained
from the IPGTT were tested using repeated measures analysis of
variance by SAS (SAS Institute Inc.; Cary, N.C.). If a significant
interaction was documented (p<0.05), values at single time
points were compared by non-paired Student's t test. All other data
were analyzed using the non-paired Student's t test: a p<0.05
was judged as significant.
[0092] Response to acute iv GLP-1 bolus. Old and young fasted
animals responded equally well to a bolus of 0.2 mmol/kg GLP-1
delivered over 30 sec. Their insulin responses were superimposable
(FIG. 1). At 2 min after completion of the bolus the insulin
response was maximal in both young (373.3.+-.43.7 pmol/l) and old
(347.7.+-.25.7 pmol/l) animals and in both groups insulin levels
had returned to baseline at 10 min.
[0093] Glucose tolerance testing. Old animals have frank glucose
intolerance when compared to young animals during an IPGTT (FIG.
2). Fasting glucose, taken just prior to the ip glucose, was not
different between treated and control animals. Blood glucose was
significantly lower during the glucose tolerance test in the
animals treated with GLP-1 when compared to control animals at the
15 (9.04.+-.0.92 vs 11.61.+-.0.23 mmol/l) and 30 (8.61.+-.0.39 vs
10.36.+-.0.43 mmol/l) min time points (FIG. 2). The old animals
were also no longer glucose intolerant when compared with young
animals. On reviewing the insulin response at the same time it can
be seen that the 15 min insulin response was significantly better
in the GLP-1-treated animals compared to the controls (FIG. 3).
Indeed, the saline-treated rats had their peak insulin level at 30
min, while the GLP-1-treated animals peaked at 15 min. This brisk
insulin response accounted for the drop in blood glucose in the
treated animals (FIG. 2). Overnight fasting levels of insulin were
higher in the GLP-1-treated animals but due to great intranimal
variation this was not statistically different from controls
(192.+-.47 vs. 134.+-.45 pmol/l). 48 hr infusion of GLP-1 in 22
month old Wistar rats potentiates insulin response to an IPGTT.
This phenomenon is observed even after termination of the GLP-1
infusion, indicating that GLP-1 is capable of inducing long-term
changes that go over and beyond modulating insulin release. The
major change in the insulin-response curve was in early insulin
release after the glucose load, and was induced by a shift in the
maximum insulin secretion from 30 min after glucose injection, as
observed in controls, to only 15 min in the GLP-1 treated rats.
[0094] Intraislet insulin content. There was variation in the
amount of insulin between the islets from each individual pancreas
as might be expected from aged animals (FIG. 4). However, there was
consistently more insulin in the islets from treated animals
(p<0.01). Islets from controls and GLP-1 treated rats had
5.31.+-.1.19 vs. 19.68.+-.3.62 ng of insulin per ug of total
pancreatic proteins, respectively.
[0095] GLP-1 plasma levels. We measured plasma GLP-1 in 3 animals 6
hrs after commencement of the GLP-1 infusion to insure both that
steady state GLP-1 levels were reached and to verify that the
peptide was actually being infused. Plasma GLP-1 level at 6 hrs was
106.7.+-.17.6 while at 48 hrs it was 125.0.+-.41.4 pmol/l. Before
the commencement of the glucose tolerance testing plasma GLP-1 was
below the level of detectability of the assay. Fasting GLP-1 levels
in control Wistar rats were 10-20 pmol/l. There was no difference
in the fasting levels of GLP-1 between the young and old animals.
Therefore, our infusion of GLP-1 raised plasma GLP-1 levels to
approximately 6 times the fasting levels. Since fed levels in
Wistar rats (Wang et al., J. Clin. Invest., 1995), as well as
humans (Gutniak et al., 1992), are reported to approximately double
after eating, the plasma levels attained with the pumps were
pharmacological.
[0096] Effect of GLP-1 on gene expression. We measured the
abundance of insulin mRNA as well as mRNA levels of other factors
involved in the early steps of glucose-mediated insulin release as
well as glucose metabolism in beta cells. Results were quantified
by densitometry, normalized by using oligo dT hybridization and
expressed in relative terms by assigning the young control result a
value of 1. FIG. 6 shows the blots for insulin mRNA from whole
pancreata in 6 young and 12 old animals ans combined results from
all the animals shown in FIG. 5. FIG. 7 shows the blots of three
isolated islet RNA preparations from old animals.
[0097] The levels of insulin mRNA were increased approximately 50%
in old vs young animals (FIG. 5A, P<0.05, and FIG. 6). GLP-1
increased the insulin mRNA in both young and old animals compared
to controls (FIG. 5A, P<0.01 and FIG. 6). Similar results can be
seen in the isolated islet preparations (FIG. 7). This increase was
entirely prevented when the animals were treated simultaneously
with Ex, an inhibitor of GLP-1 binding to its own receptor. Of
great interest is the fact that in the animals treated with Ex
alone or Ex with GLP-1, the insulin mRNA levels were lower than in
controls (p<0.05). Insulin mRNA levels fell an average of 60% in
the presence of Ex alone.
[0098] GLUT-2 mRNA levels in old animals were decreased by 70%
compared with young controls and this was entirely reversed by
GLP-1 treatment (FIG. 5B, P<0.001). The increase in GLUT2 mRNA
levels in old animals by GLP-1 can be seen in both islet (FIG. 7)
and whole pancreatic preparations (FIG. 6). in the young animals,
GLP-1 did not significantly influence the GLUT2 mRNA levels (FIG.
5). The levels fell by 50% in the presence of Ex alone (FIG. 5B,
P<0.05), but not in the animals treated with Ex and GLP-1 (FIG.
5B).
[0099] There were no differences between young and old animals in
glucokinase mRNA levels (FIG. 5C). GLP-1 significantly increased
glucokinase levels in young (FIG. 5, P<0.05), but much more so
in old animals (FIG. 5C, P<0.001, FIG. 7). Similar results were
seen in the old animals with the isolated islet preparations (FIG.
7). Ex completely prevented GLP-1-induced increases in glucokinase
mRNA.
[0100] For all preparations, the results from the pancreata were
reflected in the islets. Hexokinase I, II and III mRNA levels were
very low in the whole pancreata and islets and did not appear to be
altered by GLP-1 treatment. We also infused GLP-1 for 5 d into old
rats (n=6) and found the same results as with the 48-h
infusion.
[0101] Following continuous infusion with GLP-1, the pancreata were
surprisingly larger than control pancreata. The pancreata of
treated animals weighed 26% more than the pancreata of control
animals.
[0102] Also, surprisingly, insulin secretion remained improved even
after removal of the exogenous source of GLP-1. The biological
half-life of GLP-1's insulinotropic action in blood is 6-8 min
(Elahi et al., 1994) and since GLP-1 infusion had been terminated
at least 2 hours prior to performing the glucose tolerance testing
the continued presence of elevated GLP-1 levels, at least in the
short-term, was not necessary for the improvement in glucose
tolerance in the aging Wistar animals.
[0103] GLP-1 increases insulin biosynthesis and insulin mRNA levels
in vivo, as previously shown in insulinoma cells (Wang et al.,
Endocrinology, 1995). GLP-1 would also appear to be necessary for
the normal maintenance of mRNA levels of insulin in the pancreas.
Not only did Ex inhibit the GLP-1 effect on insulin mRNA, but it
also caused a decrease in insulin mRNA in animals given Ex alone.
Ex is a competitive inhibitor of GLP-1 binding to its receptor, a
10-fold higher concentration of Ex being required to inhibit
GLP-1's insulinotropic effect (Wang et al., J. Clin. Invest.,
1995), so presumably it was inhibiting the binding of endogenous
GLP-1 in the animals that received Ex alone. This means that GLP-1
has effects on maintaining insulin mRNA levels in the physiological
range.
[0104] It has been proposed that in Type 2 diabetes the beta cell
stores of insulin fall below a critical level, and that this causes
a subsequent reduction in glucose-induced insulin responses
(Hosokawa et al. 1996). As our data show, GLP-1 is capable of
increasing intraislet insulin content and when given continuously,
rather than just by bolus, may also induce changes beneficial to
beta cell function, over and beyond its effects on simply insulin
secretion.
Example 2
[0105] Exendin-4 is a peptide produced in the salivary glands of
the Gila Monster lizard. In the present example, we report that in
Wistar rats, bred in the National Institute of Aging (NIA), it was
a far more potent insulinotropic agent in several ways than is
GLP-1. We further report that exendin-4 leads to sustained
improvement of diabetic control in a rodent model of type 2
diabetes.
[0106] Materials. Exendin-4 and GLP-1 were purchased from Bachem
(King of Prussia, Pa.). Chemical reagents were from Sigma (St
Louis, Mo.), unless otherwise stated.
[0107] Animals. Four month old Wistar rats from the Wistar colony
in the NIA (Baltimore, Md.) were used for the acute experiments of
the effects of exendin-4 and GLP-1 (see Example 1). They had been
maintained on standard lab chow and fed ad libitum. For the
long-term experiments, diabetic mice
(C57BLKS/J-Lepr.sup.db/Lepr.sup.db) lacking the leptin receptor,
and their non-diabetic littermates were purchased at 4 weeks of age
from Jackson Laboratories (Bar Harbor, Me.). They were housed two
per cage and also were fed ad libitum. The same mice were caged
together for the duration of the study. Wistar rats are caged on
wire while the bedding for the mice was a paper based product,
"Carefresh" (Absorption Co., Bellingham, Wash.).
[0108] Protocols. Wistar rats were fasted overnight. Following
anesthesia with 50 mg/kg pentobarbital, a catheter was placed in
the femoral artery for blood sampling. A bolus of either exendin-4
or GLP-1 was given into the saphenous vein (iv) over 30 sec to 12
animals, while a bolus of normal saline (NaCl) was given to the
other six. The order of the injections was rotated. Blood (taken at
-5, 0, 2, 5, 15, 30, 60, 120, and 180 min) was drawn into
heparinized tubes containing EDTA and aprotinin for insulin
determination (See Example 1). Animals were acclimated to the
facility for at least 2 days.
[0109] Eleven diabetic and 10 non-diabetic animals received 24
nmol/kg exendin-4 ip daily thereafter (7-9 am) while 10 diabetic
and 10 non-diabetic animals received ip NaCl. Subsequently, whole
blood glucose levels, taken from a retro-orbital sinus, were
determined in the mice using a Glucometer Elite (Bayer). This
regimen was continued for 12-13 weeks. On day eight two of the
non-diabetic mice (cage-mates) and day fourteen one of the diabetic
mice died just after receiving exendin-4. Animals were weighed
weekly. After one week of the regimen blood samples were again
taken from a retro-orbital sinus for determination of insulin and
glucose levels. At the end of the regimen, fasting blood samples
were obtained for glucose and insulin levels and whole blood
containing EDTA was assayed for hemoglobin A1c (Hb A1c) on the same
day from the four groups.
[0110] In another group of eight diabetic mice 14 weeks old we gave
24 nmol/kg exendin-4 ip daily and NaCl ip daily for five days to
four each of the group.
[0111] cAMP determinations. Islets of Langerhans were harvested
from Wistar rats (Perfetti et al., 1995) and batches of 25 islets
were then incubated for 1 h at 37.degree. C. in a buffer containing
(mM) 140 NaCl, 5 KCl, 1 NaPO.sub.4 1 MgSO.sub.4, 5 glucose, 2
CaCl.sub.2, 20 HEPES, buffer (pH 7.4) and 0.1% bovine serum
albumin. Following this, they were incubated in the same buffer for
1 h in the presence of GLP-1 (1 nM) or exendin-4 (1 nM). Some
batches of islets were then washed three times with ice-cold
phosphate buffer saline (PBS) and lysed with 1 ml ice-cold 0.6 mM
perchloric acid. Other batches were washed three times in the
buffer at 37.degree. C. to remove peptide and left another 15 min
before being washed three times in ice-cold PBS followed by lysis
with the perchloric acid. The lysates (950 .mu.l) were then
transferred to microcentrifuge tubes and cAMP measured as
previously described (See Example 1) using a cAMP [.sup.3H] assay
kit (Amersham). Cellular protein was assayed using the Bradford
method (Bio-Rad, Richmond, Calif.) using bovine y-globulin as
standard.
[0112] Assays. Plasma glucose was measured by the glucose oxidase
method (Wang et al., 1997). Insulin was measured by RIA as
previously described (See Example 1). Hb A1c was assayed using a
BIO-RAD (Herculas Calif.) DiaSTAT machine which uses low pressure
cation exchange chromatography in conjunction with gradient elution
to separate hemoglobin subtypes and variants from hemolyzed blood.
The separated hemoglobin fractions were monitored by means of
absorption of light at 415 mm.
[0113] Statistical methods. All results are given as mean.+-.SEM. T
tests were based on the results of an F test that looked at the
equality of variance of the two means. If the variances were
statistically significantly different then the t test was based on
unequal variances. For determination of the EC.sub.50 the basal
plasma insulin levels were subtracted and the remaining activity at
each concentration was expressed as a percent of the maximal
activity (achieved by an excess of peptide). This was then
transformed into a logit format where logit=ln(% activity/[100-%
activity]) and was plotted as a function of the log concentration
of the compound.
[0114] Exendin-4 effects in Wistar rats. Exendin-4 was more potent
an insulinotropic agent than GLP-1 on several levels when given
intravenously. Maximal insulin response in our Wistar rats is seen
with 0.4 mmol/kg GLP-1 (De Ore et al., 1997). At this same
exendin-4 concentration maximal insulin response is approximately
doubled (FIG. 8). Insulin levels return to baseline by 10 min with
GLP-1, but with exendin-4 they actually go below baseline and have
returned to baseline by 60 min. EC.sub.50 concentration for insulin
release is lower and the maximum amount of insulin secreted by
exendin-4 is higher than with GLP-1. EC.sub.50 was 0.019 (FIG. 9)
versus 0.19 mmol/kg for exendin-4 versus GLP-1 (See Example 1),
respectively. The animals given exendin-4 had an obvious increase
in urine output for the duration of the study (we did not
quantitate the volume of urine) as they continued to urinate
frequently during the study despite the blood drawing which would
be diminishing circulating blood volume, while the GLP-1-treated
animals urinated little if any during the study.
[0115] Effects of exendin-4 and GLP-1 on cAMP levels in isolated
islets. Exendin-4 increased cAMP levels more in isolated islets
than GLP-1 at equimolar concentrations. GLP-1 increased cAMP in a
concentration-dependent manner with maximum cAMP response at 1 nM.
At that concentration of exendin-4 cAMP levels were approximately
3-fold higher (FIG. 10) than with GLP-1. This probably explains why
exendin-4 causes a higher maximal insulin release than GLP-1. In an
effort to see if exendin-4 or GLP-1 might remain on the GLP-1
receptor and so maintain an increase in cAMP after the removal of
the peptides from the buffer solution we removed the peptide from
some islets by three washes in fresh buffer and then measured cAMP
after 15 min. With both peptides, cAMP levels returned to baseline
at least by 15 min.
[0116] Effects of chronic treatment with exendin-4 in mice. The
biological activity of exendin-4, as measured by its ability to
lower blood sugar, was much longer than we expected when given ip
or subcutaneously to diabetic animals. In preliminary experiments
we found that exendin-4-treated diabetic mice had lower blood
sugars 24 hours after ip and subcutaneous (sc) injections while
with GLP-1 injections, blood sugars were back to baseline. This
lead us to design a long-term experiment with exendin-4. At the
initiation of the daily ip exendin-4 regimen in the mice fasting
blood glucose was 145.+-.51 mg/dl in the non-diabetic mice and
232.+-.38 mg/dl in the diabetic mice. After one week of treatment
the fasting glucose level in the exendin-4-treated non-diabetic
mice was 70.+-.25 mg/dl and was significantly lower than in the
NaCl-treated non-diabetic animals, 135.+-.5 mg/dl (p<0.05). The
diabetic animals had a highly significant response to exendin-4.
Glucose levels dropped to 90.+-.11 mg/dl in the exendin-4-treated
animals from 238.+-.51 mg/dl (p<0.002) in the NaCl-treated
animals (Table 1). We measured fasting insulin levels in the
diabetic animals that received NaCl or exendin-4. They were higher
in the animals that received exendin-4 (p<0.002). On the basis
of these data, we continued to treat the animals daily with
exendin-4. As the bedding was a paper-based product which turns
progressively darker with increased urination, it was clear that
the cages of the exendin-4-treated diabetic animals, while not
totally dry as were the non-diabetic cages, always were obviously
drier 24 hours after changing than the cages of the NaCl-treated
diabetic animals (FIG. 11, photograph taken after 9 weeks
treatment). We surmised that the decreased urination in the
exendin-4-treated diabetic mice was due to less osmotic diuresis
because of lower blood glucose.
[0117] The diabetic animals were clearly heavier than the
non-diabetic animals. After 9 weeks the weight of the non-diabetic
animals reached a plateau at approximately 28 grams (g) while the
diabetic animals continued to gain weight. At 13-14 weeks of
treatment the NaCl-treated animals began to lose weight (38.7 g)
while the exendin-4-treated animals maintained their weight (46.7
g).
[0118] We assayed the whole blood of the saline and
exendin-4-treated animals for Hb A1c determinations and we measured
plasma for glucose and insulin concentrations after an overnight
fast (FIG. 12). It can be seen that all these parameters were
significantly altered by the daily exendin-4 treatment. Hb A1c was
8.8% in the NaCl-treated diabetic animals vs 4.7% in the
exendin-4-treated animals (p<0.0001). Hb A1c was also lower in
the non-diabetic animals, 3.5 vs. 3.1% (p=0.0002),
exendin-4-treated vs. NaCl-treated, respectively. Glucose levels
were significantly lower (278.7.+-.30.0 vs. 517.+-.59 mg/dl,
p<0.005) and insulin levels significantly higher (4,600.+-.1,114
vs. 707.2.+-.169.7 pmol/l, p<0.02) in the exendin-4-treated
diabetic animals (FIG. 13). The trends in glucose and insulin were
the same in the non-diabetic animals with exendin-4 treatment
though not as dramatic (FIG. 13).
[0119] In the eight diabetic mice that were 14 weeks old when their
treatment began, blood sugars were 640.+-.37 mg/dl in the
NaCl-treated animals and 355.+-.21 mg/dl in the exendin-4-treated
animals after five days. Their insulin levels were 6,904.+-.705 vs.
1,072.+-.54 pmol/l, exendin-4-treated vs. NaCl-treated,
respectively.
[0120] Following sc and ip exendin-4, blood sugars in diabetic mice
had a much more prolonged biological response in that the blood
sugars stayed lower longer (up to 24 hours after an ip dose) than
was expected from the insulin response to the iv exendin-4, with
the glucose lowering effect of the ip injections being less
variable than sc. This was possibly due to greater variation in the
sc technique and perhaps even loss of the peptide during injections
in some cases.
[0121] In the studies involving ip injection of exendin-4 ip for
one week daily, the fasting blood sugars as a result of just one
injection a day were actually lower than the blood sugars of the
non-diabetic animals. It was also obvious looking at the cages each
morning which diabetic animals were receiving exendin-4 as their
cages were always drier. This effect was also seen in the
non-diabetic animals that received exendin-4.
[0122] In the experiments where exendin-4 was given daily for five
days to diabetic animals whose mean blood sugars were 640 mg/dl,
there was also a marked effect on lowering blood sugar, and insulin
levels were markedly increased. This is unlikely due to expansion
of the beta cell mass and would suggest that beta cells of diabetic
rats are still responsive to exendin-4 even in the face of such
marked hyperglycemia.
[0123] Even after 12 weeks of exendin-4, blood sugars were lower
than in the NaCl-treated animals. Typically primary antibody
responses post intraperitoneal injections take up to 14-21 days and
exendin-4 is a weak hapten at any rate. So for the first several
weeks of treatment exendin-4 would not be expected to be
neutralized by antibody. In conjunction with this, exendin-4's
biological effects are at very low concentrations and therefore
possibly the peptide has a higher affinity for the GLP-1 receptor
than for its specific antibody and so might not be totally
neutralized by antibody. Other possibilities as to why exendin-4
might not be neutralized are that there is an exendin-4-like
peptide produced in rodents that are not yet identified or that an
exendin-4-type molecule is made in utero in rodents which would
render mature animals exendin-4-tolerant.
[0124] We have given exendin-4 at a lower concentration (1 nmol/kg)
ip for one week to diabetic mice. It was just as effective at
lowering blood sugar as at the larger amount reported in this
example.
[0125] We saw no untoward effects of the daily injections on the
behavior of the mice. We have since observed that for the first 3-4
days of injection of exendin-4 the weight of the animal drops but
by the seventh day it is back to the same as NaCl-treated animals.
In our long-term study we weighed weekly and so missed the initial
drop. Except for the bedding being obviously drier each morning in
the treated diabetic animals, we could not detect any glaring
deleterious effects of exendin-4 on the animals. We therefore
suggest that exendin-4 might be superior to GLP-1 as a treatment
for Type 2 diabetes in humans.
Example 3
[0126] Using the protocol of Example 1, GLP-1 was administered by
continuous infusion for one to five days to young and old rats,
whereas control rats received comparable saline infusions.
Exendin-4, in contrast, was administered intraperitoneally one time
daily for five days according to the protocol of Example 2.
[0127] Approximately 20% of the cells in the GLP-1 treated
pancreata were positive for PCNA at five days. At the same time
point, there were proliferating cells in the islet. In addition,
there were proliferating cells lining the ducts and, more
surprisingly, in the acinar tissue, an area generally considered to
be devoid of stem cells. Also surprisingly, a number of insulin
positive cells were found outside the islets among the acinar
tissue, where insulin positive cells are not expected.
[0128] These results show that continuous infusion with GLP-1 or
repeated intraperitoneal injection with Exendin-4 for at least two
days results in an increase in total number of insulin positive
cells and in differentiation of acinar cells into insulin, IDX-1
positive cells. These results further suggest that GLP-1 and
Exendin-4 increase the proliferation of cells and, specifically, an
increase in proliferation of insulin producing cells within the
acinar tissue.
[0129] Continuous infusion of GLP-1 promotes differentiation of
non-insulin producing cells into insulin producing cells upon
contacting the non-insulin producing cells with GLP-1 for greater
than twenty-four. The effect was observed as early as 1 day, and
the maximal effect as early as seven days. Such differentiation was
surprising since the prior art showed only insulinotropic results
in beta cells. Furthermore, the present invention was surprising
because acinar cells, which have never been shown to be capable of
producing insulin, are promoted to secrete insulin upon contact
with GLP-1. The increased number of insulin producing cells remains
unchanged for at least two weeks after treatment is discontinued.
Since differentiation to an insulin producing cell is a terminal
event, de-differentiation back into a non-insulin producing cell at
even later time points is unlikely. Thus, the effect is
permanent.
[0130] Exendin-4 is shown in the present invention to have the same
effects as GLP-1 on the differentiation of insulin producing cells
from non-insulin producing cells. Surprisingly, Exendin-4 is shown
to have a much longer half-life than GLP-1. The increase in the
number of insulin producing cells, thus, can be achieved with daily
bolus injection, rather than continuous infusion, of Exendin-4 for
two days. After two injections, insulin producing cells outside the
islet are observed. The maximal effect is achieved by seven days.
As with GLP-1, the effect persists for at least two weeks, and
probably permanently, even after contact with the Exendin-4 is
discontinued.
[0131] GLP-1, Exendin-4, growth factors having amino acid sequences
substantially homologous to GLP-1 or Exendin-4, and fragments
thereof affect the differentiation of non-insulin producing cells
in vivo and in vitro. Furthermore, a variety of non-insulin
producing cells, including stem cells and acinar cells can be
promoted to differentiate into insulin producing cells. These
advances over the prior art provide methods of treating diabetes
mellitus whereby insulin producing cells are increased in number by
administration of the growth factor to a patient or by contacting
the non-insulin producing cells in vitro.
Example 4
[0132] The purpose of this study was to determine if GLP-1 and the
islet hormones, glucagon and insulin, have effects on acinar
tissue. We used the AR42J cells (Christophe, 1994), which are
derived from a rat pancreatic exocrine tumor, as a model of acinar
tissue. We then looked at some aspects of the signal transduction
system through which GLP-1 is already known to work in beta cells
(Goke et al., 1993; Holz et al., 1995; Yada et al., 1993).
[0133] Materials. GLP-1, glucagon, exendin-4 and exendin 9-39 were
obtained from Bachem (Torrance Calif.). Cholecytokinin (CCK),
insulin, genestein and vanadate were from Sigma Chemical Co (St.
Louis, Mo.). The rat pancreatic cell line, AR42J, was from American
Type Culture Collection (Rockville, Md.). Anti-tyrosine antibodies
were purchased from Upstate Biotechnology, Inc (Lake Placid,
N.Y.).
[0134] Cell Culture. AR42J cells were maintained in Dulbecco's
modified Eagle's medium (Gibco, Grand Island, N.Y.) (DMEM)
supplemented with 10% Fetal Calf Serum, 100 IU/ml penicillin, 100
.mu.g/ml streptomycin and 2 mM glutamine. Cells from passage 23-36
were used throughout this study. Cells were routinely plated at
about 10.sup.5 cells/ml in 12-well cluster dishes and incubated in
a humidified incubator at 37.degree. C. with 95% air and 5%
CO.sub.2. As AR42J cells responded poorly to CCK we routinely
incubated the cells with 10 nM dexamethasone for 48 h before use as
this was known to induce CCK responsivity in a
concentration-dependent manner (Logsdon et al., 1987).
[0135] Amylase Assay. For amylase secretion, cells were washed free
of medium with 2 ml phosphate buffered saline (PBS). Incubation was
then carried out in DMEM containing 15 mM HEPES, 0.2% bovine serum
albumin (BSA) and 0.01% soybean trypsin inhibitor. The hormones and
reagents of interest were added for 50 min at 37.degree. C. The
incubation medium was then immediately removed for amylase
determination and the cells were again washed in 2 ml ice-cold PBS.
Lysate buffer containing (in MM) 130 Tris-HCl, 10 CaCl.sub.2, 75
NaCl, and 0.2% Triton X-100 (pH 8.0) was added to the cells and the
lysates were then collected for total amylase activity (Ceska et
al., 1969). The released amylase was expressed as the percentage of
the total amylase activity of the cells.
[0136] Measurement of cAMP. Cells were grown in 12-well dishes and
treated with hormones and reagents.+-.IBMX as described above. They
were then washed 3 times in ice-cold PBS and lysed with 1 ml
ice-cold 0.6 mM perchloric acid. The lysates (950 .mu.l) were
transferred to microcentrifuge tubes and the pH adjusted to 7.0
using 5 M K.sub.2CO.sub.3. After centrifugation for 5 min at
10.sup.4 rpm, the supernatant was vacuum dried, and then recovered
in 200 .mu.l Tris/EDTA buffer. After addition of 0.15 mM
Na.sub.2CO.sub.3 (50 .mu.l) and 0.15 mM ZnSO.sup.4 (50 .mu.l),
followed by incubation for 15 min on ice, the salt precipitate was
removed by centrifugation for 15 min at 3.5.times.10.sup.3 rpm and
50 .mu.l of supernatant was assayed using a cAMP [.sup.3H] assay
kit (Amersham Corp., Arlington Heights, Ill.), (Steiner et al.,
1972). Cellular protein was measured using the Bradford method
(Bio-Rad, Richmond, Calif.) with bovine gamma-globulin as standard
(Bradford, 1976).
[0137] Measurement of intracellular calcium, [Ca.sup.2+].sub.I.
AR42J cells were loaded with the fluorescent Ca.sup.2+ probe,
indo-1 acetoxymethyl ester (indo-1/AM). The loading solution
consisted of 50 .mu.g of indo-1/AM (Molecular Probes Inc.), 30
.mu.l of dimethyl sulphoxide (DMSO) and 5 .mu.l of 25% (w/w in
DMSO) Pluronic F-127 (BASF Wyandott Corp.) This mixture was added
to 2.0 ml of cells in Hank's balance salt solution (final indo-1
concentration of 25 .mu.M) and gently mixed on a shaking plate for
1 h. The cells were then centrifuged at 400.times.g for 60 sec,
resuspended in standard bathing solution, consisting of (in mM):
137 NaCl; 5 KCl; 1.3 MgSO.sub.4; 5 CaCl; 20 HEPES; pH adjusted with
NaOH to 7.4, and stored for at least 1 h before use. Both loading
with indo-1 and the experiments were carried out at room
temperature (22-24.degree. C.). The cell suspension was placed in a
chamber on the stage of an inverted fluorescence microscope
(Spurgeon et al., 1990). The emission field was restricted to a
single cell. Indo-1 was excited at 350.+-.5 nm every 5 ms, and the
fluorescence emission was split into wavelength bands of 410.+-.5
and 490.+-.5 nm. The 410:490 fluorescence ratio (ratio F410/F490),
corrected for autofluorescence, was used as an index of
[Ca.sup.2+].sub.I, using the methodology well know in the art
(Spurgeon et al., 1990). The cell autofluorescence was assessed in
a large number of indo-1 non-loaded cells from the same batch. In a
typical experiment, the standard bathing solution was exchanged
rapidly (<200 ms) with one of the test solutions injected from a
micropipette placed in close vicinity to the cell (Janczewski et
al., 1993; Konnerth et al., 1986). Routinely, the cells were
exposed to the test solution for 240-300 s. Thereafter, the test
solution was washed out, while [Ca.sup.2+].sub.I was monitored for
additional 120-180 s. The test solutions were prepared just prior
to the experiment by adding the hormones in standard bathing
solution.
[0138] GLP-1 binding. AR42J cells were plated and cultured as
described above. At the start of the binding experiment the cells
were incubated with serum-free DMEM for 2 h at 37.degree. C. Cells
were then washed twice with 0.5 ml binding buffer containing (in
mM) 120 NaCl, 1.2 MgSO.sub.4, 13 sodium acetate, 5 KCl, 10 Tris, pH
7.6. Cells were then incubated overnight at 4.degree. C. with 0.5
ml binding buffer supplemented with 2% BSA, 500 U/ml aprotinin, 10
mM glucose, a range of concentrations of GLP-1 (0.03 nM-100 nM) and
30,000 cpm .sup.125I-GLP-1 (2,000 Ci/mmol, Peninsula, Belmont,
Calif.). We only used freshly prepared .sup.125I-GLP-1 within two
weeks of the reference date. At the end of the incubation, the
supernatant was discarded and the cells were washed three times
with ice-cold PBS. Cells were lysed with 0.5 ml 0.5 N NaOH/0.1% SDS
for 30 min at room temperature. The radioactivity was measured in
the lysates in an ICN Apec series gamma counter. Specific binding
was determined by subtracting the non-specific binding present at
500 nM GLP-1 from total binding. This method has been used
previously to characterize GLP-1 binding in CHO cells
overexpressing GLP-1 receptor and in 3T3-L1 adipocytes
(Montrose-Rafizadeh et al., J. Biol. Chem., 1997;
Montrose-Rafizadeh et al., J. Cell Physiol., 1997).
[0139] RT-PCR of the GLP-1 Receptor. Complementary DNA was
synthesized from total cellular RNA using Maloney murine leukemia
virus reverse transcriptase (Bethesda Research Laboratories,
Gaithersburg, Md.) and random hexanucleotide primer (Pharmacia LKB
Biotechnology Inc., Piscataway, N.J.). PCR amplification (30
cycles) was performed (Saiki et al., 1997) from first strand cDNA
using recombinant Taq DNA polymerase (Amplitaq, Perkin-Elmer,
Cetus). Oligonucleotide primers were on 5'- and 3'-end of the
pancreatic GLP-1 receptor sequence (Thorens, 1992), .sup.5'
ACAGGTCTCTTCTGCAACC.sup.3' and .sup.5' AAGATGACTTCATGCGTGCC.sup.3',
respectively. PCR products were then resolved on a 1% agarose gel
and visualized using ethidium bromide. The PCR products were
subcloned in pBluescript vector and sequenced using the chain
termination technique and Sequenase 2.0 kit (United States
Biochemicals, Cleveland, Ohio). The specificity of the PCR product
was also determined by the Bstx1 restriction enzyme.
[0140] Immunoprecipitation and Western blotting of the GLP-1
receptor.
[0141] AR42J cells and an insulinoma cell line, RIN 1046-38 cells,
were grown in 60 mm dishes as described above. When the cells
reached 80% confluence, they were washed twice with Krebs-Ringer
buffer containing 115 mM NaCl, 5 mM KCl, 2.5 mM CaCl.sub.2, 1 mM
MgCl.sub.2, 24 mM NaHCO.sub.3, and 25 mM HEPES and frozen in liquid
nitrogen. The frozen cells were scraped and lysed in RIPA buffer
containing 20 mM Tris-HCl: pH 8.0, 137 mM NaCl, 1% Triton X-100,
0.5% deoxycholate, 0.1% SDS, 0.2 mM PMSF, 10 .mu.g/ml leupeptin, 20
.mu.g/ml aprotinin, 1 mM Na-orthovanadate, 1 mM benzamidine.
Insoluble material was removed by centrifugation at 15,000.times.g
for 15 min at 4.degree. C. and the supernatant was collected for
immunoprecipitation and Western Blotting. Anti-GLP-1-R antibody
against the N-terminal (gift from Dr. Joel Habener, Massachusetts
General Hospital, MA) at 1:250 was added to each tube, together
with 40 .mu.l protein A and protein G. The immunoprecipitation was
carried out at 4.degree. C. overnight and the immunocomplexes were
washed twice with RIPA buffer, rewashed another two times with
washing buffer (25 mM Hepes, 0.1% Triton X-100 and 1 mM
Na-orthovanadate), then the immunocomplex pellets were solubilized
in 50 .mu.l of SDS-PAGE sample buffer at 70.degree. C. for 10 min.
The immunoprecipitated proteins were eluted with mini-resin column
and subjected to 4-20% SDS-polyacrylamide gel. After the gel was
electrotransferred to PVDF membrane, the blot was blocked with 5%
non-fat milk in TBST buffer (20 mM Tris-HCl [pH 7.5], 137 mM NaCl
and 0.1% Tween 20) for 1 h at room temperature, and then incubated
with antibody to GLP-1-receptor at 1:1500 for 1 h at room
temperature. PVDF membranes were washed three times with TBST and
incubated with horseradish peroxidase-conjugated anti-rabbit
secondary antisera for 1 h at room temperature. After a series of
washes in TBST, the blots were developed using the ECl
chemiluminescent detection system. Autoradiographs were quantified
using Image-Quant.TM. software (version 3.3) on a Molecular
Dynamics laser densitometer. In this experiment, the insulin
producing cell line RIN1046-38 cells was used as a positive control
for the presence of the GLP-1 receptor. Aliquots (20 .mu.l) of
clarified cell lysates were used to determine protein concentration
which was estimated by the Bradford method (Bradford, 1976).
[0142] Tyrosine Phosphorylation Studies. AR42J cells were
preincubated in Krebs-Ringer Balanced Buffer (KRBB) containing 115
mM NaCl, 5 mM KCl, 2.5 mM CaCl.sub.2, 1 mM MgCl.sub.2, 24 mM
NaHCO.sub.3, and 25 mM HEPES for 2 h at 37.degree. C. Then the
medium was removed and fresh KRBB was added, followed by placing
the cell on a 37.degree. C. hot plate for 5 min. After addition of
various reagents (see FIG. 24) for 5 min the reaction was
terminated by submersion of the dishes in liquid nitrogen. The
frozen cells were scraped and lysed in RIPA buffer. Insoluble
material was removed by centrifugation at 15,000.times.g for 15 min
and the supernatant was collected for immunoprecipitation and
immunoblotting. Phosphotyrosine-containing proteins from the
clarified lysates were immunoprecipitated with monoclonal
anti-phosphotyrosine antibody and separated by electrophoresis in
4-12% SDS-polyacrylamide gels under reducing conditions followed by
electrotransfer to PVDF membrane and immunoblotting with a
polyclonal anti-phosphotyrosine antibody. The blots were developed
using the ECL chemiluminescence detection system (Amersham). Total
protein content in the clarified cell lysates was assayed using the
Bradford method (Bradford, 1976).
[0143] Statistical Analysis. Where applicable results were
expressed as the mean.+-.SEM and subjected to unpaired Student's t
test. Within group comparisons were analyzed using one-way analysis
of variance (ANOVA). p<0.05 was considered statistically
significant.
[0144] Amylase release. CCK was a potent stimulus of amylase
release. Maximum stimulation was seen at 10 nM (FIG. 14). Although
glucagon (10 or 100 nM) by itself had no effect on amylase release,
when combined with CCK it inhibited, but did not fully abolish,
CCK-induced amylase release (FIG. 15; n=20, p<0.01). GLP-1 and
insulin, either alone or combined with CCK, did not influence
amylase release (FIG. 15). We also examined exendin-4
(concentrations ranging from 10 pM to 10 nM) for potential effects
on amylase release, and, similar to GLP-1, it did not appear to
influence amylase release. As GLP-1 and glucagon might be expected
to raise cAMP levels in AR42J cells we looked at the effect of
8-Bromo-cAMP (8-Br-cAMP), a cAMP analog, on amylase release to look
for specific cAMP effects. While 8-Br-cAMP appeared to have no
effect on amylase release when given alone, it reduced CCK-induced
amylase release (FIG. 16). We also used thapsigargin and ryanodine,
specific inhibitors of ryanodine receptors/ER Ca.sup.2+ release
channels and of the ER Ca.sup.2+ pumps, respectively, alone and in
combination with CCK, to investigate the role of a rise of
intracellular calcium on amylase release. The combination of
thapsigargin and ryanodine decreased, but did not fully inhibit,
CCK-induced amylase release (FIG. 17; n=3, p<0.01). NaF, which
mimics CCK's effects on amylase release in acinar tissue
(Vajanaphanich et al., 1995), did like-wise in the AR42J cells.
Genestein (300 .mu.M), the tyrosine kinase inhibitor, decreased
CCK-mediated amylase release, especially at the early time points
of the CCK treatment, while vanadate, the tyrosine phosphatase
inhibitor, increased significantly basal and CCK-mediated amylase
release (FIG. 18). We have shown that when beta cells of the
pancreas are treated with GLP-1 for 24 h there is an increase in
glucose- and GLP-1-mediated insulin release (Wang et al.,
Endocrinology, 1995). We therefore looked for any long-term effects
GLP-1 might have on amylase release. Pre-incubation of AR42J cells
for 8, 24, 48 or 72 h with GLP-1 (10 nM) and insulin (100 nM) did
not increase basal or CCK (1 nM)-induced amylase release.
[0145] [Ca.sup.2+].sub.I responses to CCK in AR42J cells. Under the
present experimental conditions, most (85%; n=35) of the AR42J
cells responded to 1 nM CCK with a transient increase in
[Ca.sup.2+].sub.I. FIG. 6A shows a representative example of the
CCK-induced [Ca.sup.2+].sub.I transients, which commenced after
5-25 secs following exposure to CCK and peaked within the next 5-15
secs. The peak [Ca.sup.2+].sub.I, assessed from the peak indo-1
fluorescence ratio (IFR), exceeded the resting IFR by 2.5-3.5 fold.
Relaxation of the [Ca.sup.2+].sub.I transients commenced
immediately following the peak and usually consisted of an initial
rapid phase, followed by a plateau and a slower final phase. After
the [Ca.sup.2+].sub.I transient, baseline [Ca.sup.2+].sub.I
decreased below the level of resting [Ca.sup.2+].sub.I, measured
prior to the exposure to CCK (FIG. 19A). During the subsequent
rest, baseline [Ca.sup.2+].sub.I showed a gradual increase, but
usually did not fully recover to the control levels within 10 min.
[Ca.sup.2+].sub.I transients elicited by a repeated exposure to CCK
prior to a full recovery of resting [Ca.sup.2+].sub.I were reduced
by 30-40% vs. the preceding [Ca.sup.2+].sub.I.
[0146] The CCK-induced [Ca.sup.2+].sub.I transients were almost
completely abolished in cells pretreated with 10 .mu.M ryanodine
and 500 .mu.M thapsigargin (FIG. 19B; n=7). These results support
the concept that in acinar cells, the ER is the major source of
changes in [Ca.sup.2+].sub.I induced by CCK (Muallem et al., 1988;
Ochs et al., 1983). Consistent with this idea, exposures to CCK
added to a nominally Ca.sup.2+-free superfusing solution (FIG. 19C)
did not appreciably affect the rate of rise or the magnitude of the
[Ca.sup.2+].sub.I transients (n=5). However, as shown in FIG. 19C,
a reduction in the extracellular Ca.sup.2+ shortened the duration
of the [Ca.sup.2+].sub.I transients, suggesting, as shown before
(Muallem et al., 1988); Ochs et al., 1983), that extracellular
Ca.sup.2+ may play a role in sustaining the delayed component of
the [Ca.sup.2+].sub.I transient initiated by CCK-induced ER
Ca.sup.2+ release.
[0147] [Ca.sup.2-].sub.I responses to GLP-1 in AR42J cells.
Exposure to GLP-1 elicited [Ca.sup.2+].sub.I responses in
approximately 50% (n=27) of AR42J cells. The GLP-1-induced
transients (FIG. 20A) displayed considerable variability, but
usually developed at a slower rate and attained smaller amplitudes
(1.5-2.5 fold increase over resting IFR) than the [Ca.sup.2+].sub.I
responses to CCK. Moreover, the GLP-1-induced [Ca.sup.2+].sub.I
transients relaxed at a slower rate than those induced by CCK
(FIGS. 20A vs. 19A and 20B,C). FIG. 20B shows the effects on
[Ca.sup.2+].sub.I of CCK applied <10 min after an exposure to
GLP-1 in the same cell. In experiments of this type, the
CCK-induced transients retained their characteristic configuration
(as in FIG. 19A) but reached smaller amplitudes. The latter effect
can be attributed, at least in part, to a reduction of the
[Ca.sup.2+].sub.I content, indicated by a reduction in the baseline
IFR, and/or partial depletion of the ER Ca.sup.2+ content (see FIG.
19). On exposure to CCK for a second time the amplitudes were even
smaller (FIG. 20C). Pretreatment with ryanodine (100 .mu.M) and
thapsigargin (500 .mu.M) virtually abolished [Ca.sup.2+].sub.I
responses to GLP-1. Taken together, these results indicate that CCK
and GLP-1 have access to the same intracellular pools of Ca.sup.2+,
presumably the ER, but perhaps release Ca.sup.2+ by differing
mechanisms. Exendin-4, the GLP-1 homolog from Gila monster, had
identical effects as GLP-1 on [Ca.sup.2+].sub.I but was
approximately one order of magnitude more potent. The GLP-1
antagonist, exendin 9-39 (Goke et al., 1993), inhibited
GLP-1-induced calcium transients when used at a 10-fold higher
concentration than GLP-1.
[0148] Effects of glucagon and 8-bromo-cAMP on [Ca.sup.2+].sub.I
AR42J cells. Exposures to glucagon (10 nM) induced
[Ca.sup.2+].sub.I responses in 70% (n=12) of AR42J cells. The
[Ca.sup.2+].sub.I transients commenced briefly after exposure to
glucagon, developed at a relatively slow rate, peaked at 200-250%
of the resting IFR level and showed a prolonged, slow relaxation
(FIG. 21A). The [Ca.sup.2+].sub.I transients induced by CCK shortly
after treatment with glucagon (or with both treatments added
simultaneously) showed an attenuated rate of rise and a very slow
rate of relaxation (FIG. 21B). Similarly, brief (60-300 sec)
exposures to 0.1 .mu.M 8-bromo-cAMP, a membrane-permeable form of
cAMP, usually did not markedly affect the rate of rise of the
CCK-induced [Ca.sup.2+].sub.I transients but markedly slowed their
rate of relaxation (FIG. 21C). A reduction in intracellular
mobilization of [Ca.sup.2+].sub.I with the acetoxymethyl ester of
dibutyryl cAMP in the presence of CCK has previously been shown in
acinar cells (Kimura et al., 1996).
[0149] GLP-1 Binding. Specific .sup.125I-GLP-1 binding, as
determined by displacement of total binding by the presence of 500
.mu.M cold GLP-1, was 0.64.+-.0.16% (n=9, the amount of specific
binding was significantly greater than zero, p<0.01) of total
radioactivity added and 27.+-.3.2% (n=9) of total binding. Because
of the low specific binding, a full Scatchard analysis was not
performed.
[0150] cAMP levels. Intracellular cAMP levels were not altered in
AR42J cells by 1 h treatment with GLP-1 (0.1 to 100 nM) or IBMX
(100 nM) in the presence or absence of CCK (1 nM), or with CCK (0.1
to 100 nM) alone. While IBMX caused a slight increase in cAMP
levels, in 3 experiments it was not statistically different from
non-IBMX-treated cells. Glucagon (10 nM) caused a 2-fold increase
in cAMP levels in the presence and absence of CCK (FIG. 22).
Exendin-4 (0.1 to 10 nM) did not alter cAMP levels.
[0151] RT-PCR of the GLP-1 Receptor. The presence of GLP-1 receptor
mRNA was detected in AR42J cells by using RT-PCR. FIG. 23 shows
that using primers identical to the known pancreatic GLP-1 receptor
sequence (Thorens, 1992), PCR product of predicted size (bp928; see
Egan et al., 1994) can be detected in AR42J cells and rat pancreas,
but not in PCR of water control. The absence of any genomic DNA
contamination is established as our primers span intronic sequences
that would yield PCR bands of 1.8 K bases. No additional bands were
observed corresponding to contaminating genomic DNA PCR in our PCR
reactions. The PCR reactions were cloned, partially sequenced and
identified to be the beta cell GLP-1 receptor.
[0152] Western blot analysis of GLP-1 expression. Using an antibody
against the N-terminal region of the GLP-1 receptor, specific bands
were obtained at 65 and 45 kDa in the positive control cells, the
RIN1046-38 cells, and in the AR42J cells. These have been shown to
correspond to the mature and core-glycosylated GLP-1 receptors,
respectively (FIG. 24).
[0153] Tyrosine Phosphorylation Studies. In the absence of any
stimulation, some proteins exhibited a basal level of
phosphorylation which was increased in the presence of CCK and NaF,
but not GLP-1 (FIG. 25). Four proteins (46, 66, 120 and 190 kDa)
were the most obviously influenced in the presence of CCK with at
least a 2-fold increase in the phosphorylation levels of those
proteins. Genistein decreased tyrosine phosphorylations induced by
CCK and diminished CCK-mediated amylase release, as already shown
in FIG. 18.
[0154] AR42J cells respond in a physiological manner to CCK as
evidenced by induction of amylase release in a
concentration-dependent manner and increased intracellular calcium.
CCK also induced protein tyrosine phosphorylation as had previously
been shown (Lutz et al., 1993). CCK induced substantial increases
in tyrosine phosphosubstrates of kDa 190, 120, 66 and 46 on the
basis of apparent molecular masses when separated on
SDS-polyacrylamide gels. Two of those phosphorylations, 120 and 66
kDa, have already been described (Id.). Inhibition of tyrosine
phosphorylation by genistein inhibited amylase release and also
decreased tyrosine phosphorylation events. This suggests that in
AR42J cells, as in acinar cells, that tyrosine phosphorylation is
involved in regulated amylase secretion. Insulin induced
phosphorylation of most probably its own receptor beta subunit at
97 kDa. NaF, a well known activator of G proteins (Rivard et al.,
1995), has previously been shown to mimic CCK's effects in acinar
cells in that it increases amylase release and increases tyrosine
kinase activity in acinar cells (Id.). NaF mimics CCK's effects on
tyrosine phosphorylation events in AR42J cells and therefore lends
credence to the hypothesis that there exists a fluoride-sensitive G
protein that functions as a transducer between the CCK receptor and
tyrosine phosphorylation (Id.).
[0155] GLP-1 clearly increased intracellular calcium but did not
appear to increase amylase release alone or with CCK in AR42J
cells. No increase in cAMP was demonstrated in the presence of
GLP-1 though it was obvious with glucagon. Malhotra et al. (1992),
using rat acinar cells, stated that exendin-4, the Gila monster
venom that is homologous to GLP-1, potentiated CCK-induced amylase
release and increased cellular cAMP but did not discuss GLP-1
effects. However, increased cAMP was not seen until 10.sup.-8 M
exendin-4 was used, at which concentration exendin-4 may be
interacting through other receptors (Id.). Likewise the effect on
potentiating CCK-induced amylase release (from 12% of total amylase
released by CCK alone vs. 16% with exendin-4 and CCK together) was
seen with 10.sup.-8 M exendin-4 and reached statistical
significance only at the 15 min time point (p<0.02) of a time
course of exposure to CCK for 1 hour. The methods may not be
sensitive enough to pick up such a very small and time-specific
effect of GLP-1 or exendin-4 if it were occurring, and, once again,
the effect on secretion shown by Malhotra et al. may be due to
interaction with other receptors. In beta cells of the pancreas
exendin-4 increases cAMP and insulin secretion with concentrations
as low as 10.sup.-10M concentrations (Goke et al., 1993).
Alternatively, due to low receptor affinity, small, acute changes
in cAMP levels with GLP-1 may not have been detected.
[0156] The response of AR42J cells is similar to that seen in
peripheral cells (liver, fat, and skeletal muscle), which do not
show an increase in cAMP levels either (Valderde And
Villanueva-Penacarrillo, 1996). It appears that GLP-1 might be
coupled to either a different G-protein subtype than in beta cells
or to other G-protein subtypes. The CCK receptor has been shown to
be coupled to G.sub.i subtypes as well as G.sub.q subtypes in
acinar cells (Schnefel et al., 1990). In AR42J cells, GLP-1 may be
coupled to at least a G.sub.i subtype and possibly other G-protein
alpha subunits. In 3T3-L1 adipocytes, in which GLP-1 increases
lipid synthesis and glucose uptake, it has been shown that the
GLP-1 receptor is most likely coupled to a G.sub.i subtype
(Montrose-Rafizadeh et al., J. Biol. Chem., 1997) and that in CHO
cells which overexpress the GLP-1 receptor it is coupled to other
alpha subunits (Montrose-Rafizadeh et al., Diabetes, 1997).
[0157] Similar to CCK, the rise in intracellular calcium induced by
GLP-1 was from the endoplasmic reticulum. However, the pattern of
the calcium gradients was not the same as with CCK, implying that
the signaling to the release of calcium by CCK was possibly
different from that by glucagon and GLP-1. GLP-1 did not increase
tyrosine phosphorylation events. This demonstrates once again the
importance of tyrosine phosphorylation for regulated amylase
release. It also demonstrates that pathways independent of an
elevation of intracellular calcium are important for the secretion
of amylase. This is further underscored by the results obtained in
the presence of thapsigargin and ryanodine. While they prevented
any rise in intracellular calcium they reduced, but did not
completely prevent, CCK-induced amylase release. So a rise of
intracellular calcium is necessary for the full expression of
CCK-induced amylase release but of itself it is clearly not
sufficient to induce amylase release in AR42J cells.
[0158] Any cell type may contain diverse beta subunits of the
GTP-binding proteins (von Weizsacker et al., 1992). This could mean
that depending on the subtype activated, i.e., G.sub.q by CCK or
GLP-1, G.sub.s by glucagon or GLP-1, or G.sub.i by both CCK and
GLP-1, a different G.sub..beta.y subunit may be released. A
specific G.sub..beta.y might then be required for the tyrosine
phosphorylation events observed in AR42J cells as already described
for mitogen-activated protein kinase activation (Hawes et al.,
1995). It also raises the possibility that if two different
G.sub..beta.y subunits are released by the action of one hormone
they might have additive or antagonistic effects on various
down-stream events.
[0159] GLP-1 receptors are present on AR42J cells. Their activation
by GLP-1 and exendin-4 leads to increased intracellular calcium,
probably from the ER. Their activation, however, does not lead to
an increase in amylase release and CCK-induced amylase release is
not potentiated.
Example 5
[0160] As discussed in Example 4, GLP-1 receptors are present on
AR42J cells, and acute treatment of AR42J cells with GLP-1 raises
intracellular calcium in the cells. Furthermore, previous studies
showed that, although dexamethasone promoted AR42J cells to become
acinar-like cells (Christophe, 1994), betacellulin and activin A
converted approximately 10% of AR42J cells into insulin-producing
cells (Mashima et al., J. Clin. Invest. 1996). Similarly, after
exposure to hepatocyte growth factor (HGF, also known as heptocyte
scatter factor (HSF)), about 3% of AR42J cells were insulin
positive; whereas exposure to HGF and activin A resulted in about
10% insulin positive cells (Mashima et al., Endocrinology, 1996).
There was no mention in either of the above studies relating to
either GLP-1 or exendin-4. Furthermore, either GLP-1 or Exendin-4
can convert AR42J cells into insulin-producing cells in far greater
numbers than combined treatment with activin A and betacellulin or
combined treatment with HGF and activin A. The mechanism of the
effect by GLP-1 or exendin-4 may involve, as a final step,
activation of the ERK/MAPK pathway, as inhibition of ERK activation
prevented the insulin and glucagon production.
[0161] Materials. AR42J cells were obtained from ATCC (Rockville,
Md.). GLP-1, exendin-4 and exendin 9-39 (the GLP-1 receptor
antagonist) were from Bachem (Torrance, Calif.). Anti-insulin and
anti-glucagon antibodies were from Linco (Charles, Mo.). Anti-rat
ERK1/2 antibody (ERK1-CT) and Myelin Basic Protein (MBP) were
purchased from Upstate Biotechnology Incorporated (Lake Placid,
N.Y.). Insulin radioimmunoassay reagents were from Peninsula
Laboratories (Belmont, Calif.). Protein measurement reagents were
obtained from Bio-Rad (Hercules, Calif.). Peroxidase ABC kits were
obtained from Vector Laboratories (Burlingame, Calif.). Tian.TM.
One Tube RT-PCR system was purchased from Boehringer Mannheim
(Indianapolis, Ind.). Deoxyribonuclease I was obtained from Gibco
BRL (Gaithersburg, Md.). Glass coverslips were from VWR Scientific
(Baltimore, Md.). The protein kinase C (PKC) inhibitor
1-o-Hexadecyl-2-o-methyl-rac-glycerol (PKI), and the MAP kinase
kinase (MAPKK) inhibitor, PD98059, were from Calbiochem (San Diego,
Calif.).
[0162] Cell culture. AR42J cells were maintained in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal calf
serum (FBS), 100 IU/ml penicillin, 100 .mu.g/ml streptomycin and 2
mM glutamine. Cells from passage 23-35 were used throughout this
study. Cells were routinely plated at a density of about 10.sup.5
cells/ml in 12-well cluster dishes or on coverslips and incubated
in a humidified incubator at 37.degree. C. with 95% air and 5%
CO.sub.2.
[0163] Immunocytochemistry analysis. Cells were cultured on glass
coverslips, washed with phosphate buffered saline (PBS) to remove
serum, and fixed with 0.5% glutaraldehyde in PBS. Cells were
permeabilized with 0.2% Triton X-100 for 5 min and the rest of the
procedure was carried out at room temperature in a humidified
chamber. Suction was used to remove reagents between each step but
drying of specimens was avoided. Sufficient reagent was used to
cover each specimen (approximately 1 or 2 drops was usually
adequate). The coverslips were incubated in 0.3% H.sub.2O.sub.2 in
PBS for 30 min to quench endogenous peroxidase activity and washed
in PBS.times.3 times, followed by incubation with 2% goat serum in
PBS for 30 min to block non-specific binding of IgG. Excess serum
was removed by blotting. The specific primary polyclonal antisera
(anti-insulin 1:300; anti-glucagon 1:300) were used. Antibody was
diluted in PBS containing 1% goat serum. This was applied to the
coverslip and incubated at room temperature for 1 h. The coverslips
were washed.times.3 times in PBS, each time for 5 min, then
incubated with biotinylated second antibody for 1 h and
washed.times.3 times with PBS. Avidin-biotin-peroxidase complex in
PBS was applied for 30 min. Immunoperoxidase labeling was performed
with a Vectostain ABC kit (Vector Labs, Burlingame, Calif.). After
extensive washing in PBS for 4-5 times (each 5 min), the coverslips
were incubated in diaminobenzidine tetrahydrochloride (DAB) in PBS,
with 0.01% hydrogen peroxide for 3 min. The reaction was stopped by
washing the coverslips in PBS and examined under a light
microscope. To confirm specific staining, samples incubated with
preabsorbed primary antibody were used as negative control, and the
insulin producing cell line RIN 1046-38 cells were used as positive
control for our experiments. The avidin-biotin-peroxidase (ABC)
procedure was performed according to methods known in the art (Hsu
et al., 1981).
[0164] Measurement of immunoreactive insulin. AR42J cells were
cultured as before in 12-well cluster plates. When cells reached
60% confluence, they were treated with GLP-1 for 3 days. At the
beginning of the experiments, an aliquot of the medium was taken in
order to assay the insulin accumulation in the medium. Then the
cells were twice washed with Kreb's Ringer Balanced Buffer (KRBB)
and incubated in the same buffer containing 10 mM glucose for
another 1 h. The medium was collected and kept at -20.degree. C.
until insulin levels were assayed by RIA (See Example 1; Wang et
al., Endocrinology, 1995). The cells were washed with PBS and
detached with 0.25% typsin and 0.02% EDTA. The cell pellet was
collected and lysed with formic acid for protein determination by
the Bradford method (Bradford, 1976), using bovine--globulin as
standard.
[0165] Reverse-transcription polymerase chain reaction (RT-PCR).
Total RNA was isolated from treated AR42J cells by the methods of
Chomczynski and Sacchi (1987). The total RNA samples were
pretreated with DNAse in 20 mM Tris-HCL (pH 8.4), 2 mM MgCl.sub.2
and 50 mM KCl to remove any traces of contaminating genomic DNA.
RT-PCR was undertaken in a volume of 50 .mu.l of buffer containing
50 mM KCl, 10 mM Tris-HCl, 3.5 mM MgCl.sub.2, 200 .mu.M each dNTPs,
0.4 .mu.M each of sense and antisense primers to rat insulin I and
II (insulin sense primer=5'TGCCCAGGCTTTTGTCAAACAGCACCTT3'; insulin
antisense primer=5'CTCCAGTGCCAAGGTCTGAA 3'). Amplification was
undertaken for 25 cycles at denaturing temperature 94.degree. C.
for 1 min, annealing temperature 60.degree. C. for 45 sec and an
extension temperature 72.degree. C. for 1 min. mRNA from RIN
1046-38 cells were used as a positive control. In the case of
glucagon RT-PCR, the denaturing and extension temperature was
similar to insulin except the annealing temperature was 65.degree.
C. for 1 min (glucagon sense primer=5'
GTGGCTGGATTGTTTGTAATGCTGCTG3'; antisense primer=5'
CGGTTCCTCTTGGTGTTCATCAAC3'). The RT-PCR products were visualized by
ethidium bromide staining on 2% agarose gels.
[0166] MAP Kinase Activity. After treatment, 60 mm dishes of 80%
confluent cells were lysed at 4.degree. C. in lysis buffer (in mM):
50 TRIS-HCl, PH 8, 150 NaCl, 5 EDTA, 1% NP-40, 0.25% sodium
deoxycholate, 1 NaF, 10 sodium pyrophosphate, 0.1 PMSF, 1 sodium
orthovanadate, 20 .mu.g/ml aprotinin, and 10 .mu.g/ml leupeptin.
The cell lysate was clarified by centrifugation at 16,000.times.g
at 4.degree. C. for 20 min. The clarified cell lysate was
immunoprecipitated overnight at 4.degree. C., rotating with 4.5
.mu.g of ERK1-CT antibody and 40 .mu.l of packed protein G+protein
A agarose resin (Oncogene Research Product, Cambridge, Mass.). The
immune pellet was assayed for MAPK activity using MBP as the
substrate. MBP (18.6 .mu.g) was phosphorylated at 20.degree. C. for
10 min in a final volume of 60 .mu.l containing 20 mM Hepes, PH
7.4, 10 mM MgCl.sub.2, 1 mM DTT, 20 uM unlabeled ATP and 40 .mu.Ci
(3,000 Ci/mmol) [.sup.32P]-ATP. The reaction was terminated by the
addition of 25 .mu.l of 3.times. Laemmli sample buffer and heating
at 70.degree. C. for 10 min. MAPK activity was assessed by SDS-PAGE
and auto-radiography. The autoradiograms were quantified by
densitometry.
[0167] Amylase Assay. For amylase determination, cells were washed
free of medium with 2 ml PBS. Incubation was then carried out in
DMEM containing 15 mM HEPES, 0.2% BSA and 0.01% soybean trypsin
inhibitor. CCK (1 nM) was added for 50 min at 37.degree. C. The
incubation medium was then immediately removed for amylase
determination and the cells were again washed in 2 ml ice-cold PBS.
Lysate buffer containing (in mM) 130 Tris-HCl, 10 CaCl.sub.2, 75
NaCl, and 0.2% Triton X-100 (pH 8.0) was added to the cells and the
lysates were then collected for total amylase (Ceska et al., 1969).
The released amylase was expressed as the percentage of the total
amylase in the cells.
[0168] Statistics. All data values are shown as mean.+-.SEM, and
the differences among the treated groups were analyzed by one
factor ANOVA analysis. Differences between treated and non-treated
cells were analyzed using the Students' t test. p<0.05 was
considered significant difference.
[0169] Effects of GLP-1 on the expression of insulin and glucagon.
After GLP-1 or exendin-4 treatment, AR42J cells convert to
insulin-containing cells. Using anti-insulin antibody, intense
immunostaining was present in AR42J cells. In contrast, no
immunostaining was observed in AR42J cells not treated with GLP-1.
Preabsorption of the antibodies with an excess of insulin and
glucagon prevented staining (FIG. 26).
[0170] With 1 nM GLP-1, .about.10% converted into insulin-positive
cells after 3 days. When 10 nM GLP-1 or 0.1 nM exendin-4 were used
for 3 days, .about.25% of the AR42J cells converted into
insulin-positive cells. In some areas of the slides whole sheets of
contiguous cells became positive for insulin. An occasional
glucagon-positive cell appeared as early as 24 h. By 48 h, 20% of
all treated AR42J cells were glucagon positive, with .about.6% of
the cells being insulin-positive. By 72 h fully half of all the
treated cells contained glucagon. The number of cells contained
glucagon declined thereafter but still .about.25% of cells remained
insulin-positive for at least 7 days (FIG. 27). The presence or
absence of dexamethasone in the culture medium did not in any way
influence the number of cells that converted to "endocrine" cells
in the presence of GLP-1. When PD98059 (50 .mu.M), a selective
inhibitor of MEK which phosphorylates and activates ERK, or PKI
(300 .mu.M) were added concurrently with GLP-1, conversion of the
cells did not occur.
[0171] Insulin release. After a 3 day period of treating AR42J
cells with 1 nM GLP-1, insulin was readily detected in the culture
medium by radioimmunoassay. Over 3 separate cultures 5.1.+-.0.4 pg
insulin/.mu.g protein (mean.+-.SD) was present in the cell culture
medium from the 60-72 h time period. To investigate whether glucose
could induce insulin secretion from 3 day-GLP-1-treated and
non-treated cells, the medium was removed and the cells washed with
glucose-free KRBB.times.3 times. This was followed by the addition
of KRBB containing 10 mM glucose for 1 h and the cells maintained
at 37.degree. C. The incubating buffer was collected and insulin
measured. Insulin was zero from the control cells, whereas there
was 0.65.+-.0.15 pg insulin/.mu.g protein present in the buffer of
cells that had previously seen 3 days of GLP-1. Insulin secretion
was barely detected in the presence of 200 .mu.M PKI or in the
presence of PD98059 (50 .mu.M).
[0172] RT-PCR analysis. RT-PCR analysis demonstrated a 187 bp rat
insulin I and II mRNA in GLP-1-treated AR42J cells for 3 days. The
RIN cells were used as positive control. In this experiment, RNA
was pretreated with DNAse, only a mRNA fragment of insulin I and II
with the predicted length was amplified, thus the band that
appeared at 187 bp was the specific insulin mRNA product (FIG.
28A). In contrast, no RT-PCR products were detected in the negative
control or in non-GLP-1-treated cells. Northern blot analysis of
GLP-1-stimulated AR42J cells was faintly positive and therefore the
band scanned poorly. Glucagon mRNA at 236 bp was detected in
GLP-1-treated AR42J cells at 48 h (FIG. 28B).
[0173] AMP kinase activity ERK activation was readily detected in
AR42J cells. Its activity was markedly increased with GLP-1 and
exendin-4, the Gila monster venom peptide, that is 52% homologous
to GLP-1 and been shown to be an insulin secretagogue (Goke et al.,
1993). Exendin-4 was about 100-fold more potent than GLP-1 (FIGS.
29A and 29B). PKI (300 .mu.M) alone decreased MAPK activity to less
than that of control cells.
[0174] Amylase change. Incubating AR42J with dexamethasone for 72 h
increased amylase content 6.6-fold (12.57 U/l) in the cells
compared with non-dexamethasone-treated cells (1.88 U/l). When
GLP-1 was added together with dexamethasone, the total amylase
content was decreased compared to dexamethasone treatment alone
(7.76 U/l). The acute response to CCK (1 nM) was also decreased in
the cells that were pretreated with GLP-1 for 72 h (FIG. 30).
[0175] GLP-1 induces AR42J cells to differentiate into pancreatic
endocrine cells, or, at the very least, into cells with endocrine
traits. In conjunction with this observation, the same pattern
occurs in the developing embryonal pancreas (Guz et al., 1995).
Glucagon is the first hormone detected (Rall et al., 1973). It is
postulated that cells containing glucagon are precursor cells for
various other types of islet endocrine cells, and that they, in
turn, arose from ductal epithelium (Guz et al., 1995). But the
mechanisms regulating formation and differentiation of the
pancreatic hormone-producing cells is still largely undetermined.
GLP-1 turns on glucagon production very early in AR42J cells and
this is then closely followed by insulin production. Eventually the
majority of the "endocrine" AR42J cells are insulin-producing cells
as the glucagon production wanes. Exendin-4 was even more potent
than GLP-1 as a factor for insulin production in AR42J cells. Some
insulin-containing cells were seen in the presence of
concentrations as low as 10.sup.-11 molar exendin-4. GLP-1 (and/or
a GLP-1-like peptide, perhaps resembling exendin-4) may be a
differentiation factor in the embryo for islets. Such a peptide
would be expected to be present in high concentrations locally as
the pancreas is forming from the primitive gut.
[0176] Glucagon has been hypothesized to be the signal for
differentiation of the beta cells by increasing cAMP which would
lead to a decrease in cell proliferation and to changes in
macromolecular synthesis, culminating in the beta cell phenotype.
(Rall et al., 1973) This might still be applicable in the AR42J
cells. As glucagon is the hormone seen first in our system it could
be the signal for insulin production. GLP-1 produced in the
primitive gut might be the signal for the glucagon expression (and
subsequently the insulin expression), which would lead to the
further formation of endocrine cells and islet-like structures.
[0177] The final common pathway to "endocrine" cell differentiation
in AR42J cells, as well as other cell types, is likely through the
ERK/MAPK pathway. GLP-1 or Exendin-4 produce little or no insulin
staining in AR42J cells and no insulin into the medium when the ERK
activity is inhibited. Little or no insulin in the presence of
GLP-1 and a PKC inhibitor is observed. As the GLP-1 receptor is
known to be G-protein linked, is present on AR42J cells, and raises
intracellular calcium in AR42J cells (see Example 4), its
activation by ligand binding probably leads to PKC activation as
well as other as yet undetermined down-stream events (Nishizuka,
1984; Zamponi et al., 1997). PKC, in turn, has been shown to be one
of the factors that activates the MAPK pathway (Offermanns et al.,
1993; Siddhanti et al., 1995). Therefore, blocking PKC activation
by GLP-1 probably lead to diminishing MAPK activity and prevented
the development of the "endocrine" cell phenotype.
[0178] Moreover, not all cells convert to "endocrine" cells with
GLP-1, even with incubations as long as 7 days. The treated AR42J
cells possess both exocrine and neuroendocrine properties, as has
been described for untreated AR42J cells (see Christophe, 1994).
Morphologically, various populations of the treated cells do not
appear the same. Thus, sub-populations of cells may be present in
the untreated AR42J cells. Specifically, some of these populations
may possess the GLP-1 receptor, and others may not. Cell
preparations made from the total population of AR42J cells possess
GLP-1 receptors by Western blotting, PCR analysis and partial
sequencing. On sequencing, the receptor is identical to that found
on beta cells and which has been fully characterized already (see
Example 4). Furthermore, at least 50% of the AR42J cells increase
intracellular calcium in response to GLP-1. Therefore, GLP-1
probably activates a series of events which require increased
intracellular calcium and as yet other hitherto unknown factors
which are definitely present in AR42J cells and commit them to
become "endocrine" cells.
Example 6
[0179] Subjects diagnosed with Type 1 diabetes can be selected for
treatment with GLP-1 or exendin-4. The treatment method of Gutniak
et al., 1992 can be modified so that GLP-1 is administered for at
least twenty four hours by cannula in the antecubital vein. The
cannula can be connected to an insulin infusion system pumping
between 0.03 and 4.80 nmoles/kg/min GLP-1. Blood glucose levels can
be monitored regularly, using methods well known in the art, during
administration of GLP-1 and following the twenty-four hour period
of GLP-1 administration. After twenty four hours of GLP-1 infusion,
the subject shows levels of blood glucose that approach normal
levels and has a reduced need for insulin therapy.
[0180] Alternatively, subjects with Type 1 diabetes can be treated
with exendin-4 by a single subcutaneous injection or by daily
repeated subcutaneous injections of 0.01 nmole/kg to 0.4 nmole/kg.
Blood glucose levels can be monitored regularly after
administration of exendin-4. The need for insulin replacement
therapy should decrease and blood glucose levels should approach
normal levels.
[0181] The preceding examples are intended to illustrate, but not
limit, the invention. While they are typical of those that might be
used, other procedures known to those skilled in the art may be
alternatively employed.
[0182] By contacting cells with GLP-1 or Exendin-4 as described
above, it is understood that both GLP-1 or Exendin-4, substantially
homologous sequences, or fragments thereof could be used
together.
[0183] Throughout this application various publications are
referenced. The disclosures of these publications in their entirety
are hereby incorporated by reference into this application in order
to more fully describe the state of the art to which this invention
pertains.
REFERENCES
[0184] 1. Arver et al. 1991. Different aetiologies of type 2
(non-insulin-dependent) diabetes mellitus in obese and non-obese
subjects. Diabetologia 34: 483-487. [0185] 2. Bradford. 1976. A
rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye
binding. Annal. Biochem. 72: 248-254. [0186] 3. Bressler, R. and
Johnson, G. G. 1997. Pharmacological regulation of blood glucose
levels in non-insulin dependent diabetes. Arch. Int. Med.
157:836-848. [0187] 4. Busik et al. 1997. Glucose-Specific
Regulation of Aldose Reductase in Capan-1 Human Pancreatic Dcust
Cells In Vitro. J. Clin. Invest. 100: 1685-1692. [0188] 5. Ceska et
al. 1969. A new and rapid method for the clinical determination of
alpha-amylase activities in human serum and urine. Clin. Chim. Acta
26: 437-444. [0189] 6. Chen and Drucker. 1997. Tissue-specific
expression of unique mRNAs that encode pro-glucagon-derived
peptides or exendin-4 in the lizard. J. Biol. Chem. 272: 4108-4115.
[0190] 7. Chigwin et al. 1979. Isolation of biologically active
ribonucleic acid from sources enriched in ribonuclease.
Biochemistry. 18: 5294-5299. [0191] 8. Chomczynski and Sacchi.
1987. Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Analyt. Biochem. 162:
156-159. [0192] 9. Christophe. 1994. Pancreatic tumoral cell line
AR42J: an amphicrine model. Am. J. Physiol. 266: G963-971. [0193]
10. De Ore et al. 1997. The effects of GLP-1 on insulin release in
young and old rats in the fasting state and during an intravenous
glucose tolerance test. J. Geront. 52: B245-249. [0194] 11. Drucker
et al. 1987. Glucagon-like Peptide 1 stimulates insulin gene
expression and increases cyclic AMP in a rat islet cell line Proc.
Natl. Acad. Sci. USA. 84: 3434-3438. [0195] 12. Egan et al. 1994.
Glucagon-like peptide-1 (7-36) amide (GLP-1) enhances
insulin-stimulated glucose metabolism in 3T3-L1 adipocytes: one of
several potential extrapancreatic sites of GLP-1 action.
Endocrinology 135: 2070-2075. [0196] 13. Egan et al. 1991. Glucose
stimulated insulin release by individual beta cells: potentiation
by glyburide. J. Exp. Med. Biol. 196: 203-210. [0197] 14. Elahi, et
al. 1994. The insulinotropic actions of glucose-dependent
insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (737)
in normal and diabetic subjects. Regulatory Peptides. 51: 63-74.
[0198] 15. Elahi, et al. 1985. The effect of age and glucose
concentration on insulin secretion by the isolated perfused
pancreas. Endocrinology 116: 11-16. [0199] 16. Fehmann and Habener.
1992. Insulinotropic hormone glucagon-like peptide-1 (7-37)
stimulation of proinsulin gene expression and proinsulin
biosynthesis in insulinoma .beta.TC-1 cells. Endocrinology 130:
159-166. [0200] 17. Fehmann et al. 1995. Cell and Molecular Biology
of the Incretin Hormones Glucagon-Like Peptide-I and
Glucose-Dependent Insulin Releasing Polypeptide. Endocrine Rev.
16:390-410. [0201] 18. Ghazzi et al. 1997. Cardiac and glycemic
benefits of troglitazone treatment in NIDDM. Diabetes. 46: 433-439.
Care. 15: 270-276. [0202] 19. Glisin et al. 1974. Ribonucleic acid
isolated by cesium chloride centrifugation. Biochemistry. 13:
2633-2637. [0203] 20. Goke et al. 1993. Exendin-4 is a potent
agonist and truncated exendin-(9-39)-amide an antagonist at the
GLP-1-(7-36)-amide receptor of insulin-secreting .beta.-cells. J.
Biol. Chem. 268; 19650-19655. [0204] 21. Goldfine et al. 1997. The
Endocrine Secretion of Human Insulin and Growth Hormone by Exocrine
Glands of the Gastrointestinal Tract. Nature Biotechnology
15:1378-1382. [0205] 22. Gromada et al. 1998. Glucagon-Like Peptide
1(7-36) Amide Stimulates Exocytosis in Human Pancreatic
.beta.-Cells by Both Proximal and Distal Regulatory Steps in
Stimulus-Secretion Coupling. Diabetes 47:57-65. [0206] 23. Gutniak
et al. 1992. Antidiabetogenic effect of glucagon-like peptide-1
(7-36) amide in normal subjects and patients with diabetes
mellitus. N. Engl. J Med 326:1316-1322. [0207] 24. Guz et al. 1995.
Expression of murine STF-1, a putative insulin gene transcription
factor, in .beta. cells of pancreas, duodenal epithelium and
pancreatic exocrine and endocrine progenitors during ontogeny.
Development 121: 11-18. [0208] 25. Hawes et al. 1995. Distinct
pathways of G.sub.i- and G.sub.q-mediated mitogen-activated protein
kinase activation. J. Biol. Chem. 270: 17148-17153. [0209] 26. Holz
et al. 1995. Activation of a cAMP-regulated Ca.sup.+-signaling
pathway in pancreatic beta-cells by the insulinotropic hormone
glucagon-like-peptide-1. J. Biol. Chem. 270: 17749-17757. [0210]
27. Hosokawa et al. 1996. Mechanism of impaired glucose-potentiated
insulin secretion in diabetic 90% pancreatectomy rats. Study using
glucagon like peptide-1 (7-37). J. Clin. Invest. 97: 180-186.
[0211] 28. Hsu et al. 1981. Use of avidin-biotin-peroxidase complex
(ABC) in immunoperoxidase techniques: A comparison between (ABC)
and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem.
29: 577-580. [0212] 29. Janczewski and Lakatta. 1993. Buffering of
calcium influx by sarcoplasmic reticulum during the action
potential in guinea-pig ventricular myocytes. J. Physiol. 471:
343-363. [0213] 30. Kimura et al. 1996. High concentrations of
cholecystokinin octapeptide suppress protein kinase C activity in
guinea pig pancreatic acini. Peptides 17: 917-925. [0214] 31.
Konnerth et al. 1986. Nonsynaptic epileptogenesis in the mammalian
hippocampus in vitro. I. Development of seizure like activity in
low extracellular calcium. J. Neurophysiol. 56: 409-423. [0215] 32.
Logsdon et al. 1987. Mechanism of glucocorticoid-induced increase
in pancreatic amylase gene transcription. J. Biol. Chem. 262:
15765-15769. [0216] 33. Lutz et al. 1993. A role for
cholecystokinin-stimulated protein tyrosine phosphorylation in
regulated secretion by the pancreatic acinar cell. J. Biol. Chem.
268: 11119-11124. [0217] 34. Malhotra et al. 1992. Exendin-4, a new
peptide from heloderma suspectum venom, potentiates
cholecystokinin-induced amylase release from rat pancreatic acini.
Regul Pept. 41: 149-156. [0218] 35. Mashima et al. 1996.
Betacellulin and activin A coordinately convert amylase-secreting
AR42J cells into insulin-secreting cells. J. Clin. Invest.
97:1647-1654. [0219] 36. Mashima et al. 1996, Formation of
Insulin-Producing Cells from Pancreatic Acinar AR42J Cells by
Hepatocyte Growth Factor. Endocrinology 137: 3969-3976. [0220] 37.
Matschinsky. 1990. Glucokinase as glucose sensor and metabolic
signal generator in pancreatic .beta.-cells. Diabetes. 39: 647-652.
[0221] 38. Montrose-Rafizadeh et al. 1997. High potency antagonists
of the pancreatic glucagon-like peptide-1 receptor. J. Biol. Chem.
272: 21201-21206. [0222] 39. Montrose-Rafizadeh et al. 1994.
Incretin hormones regulate glucose-dependent insulin secretion in
RIN 1046-38 cells: mechanism of action. Endocrinology. 135:
589-594. [0223] 40. Montrose-Rafizadeh et al. 1997. Evidence of
direct coupling of pancreatic GLP-1 receptor to different G-protein
alpha subunits. Diabetes 46: 0724a (Abstr.) [0224] 41.
Montrose-Rafizadeh et al. 1997. Novel signal transduction and
peptide specificity of glucagon-like peptide receptor in 3T3-L1
adipocytes. J. Cell. Physiol. 172: 275-280. [0225] 42. Muallem et
al., 1988. Agonist-sensitive calcium pool in the pancreatic acinar
cell. I. Permeability properties. Am. J. Physiol. 255: G221-228.
[0226] 43. Mueckler. 1990. Family of glucose-transporter genes.
Implications for glucose homeostasis and diabetes. Diabetes. 39:
6-11. [0227] 44. Nathan et al. 1992. Insulinotropic action of
glucagon like peptide-1-(7-37) in diabetic and nondiabetic
subjects. Diabetes Care. 15: 270-276. [0228] 45. Nauck et al. 1993.
preserved incretin activity of Glucagon-like peptide 1 (7-36) amide
but not of synthetic human gastric inhibitory polypeptide in
patients with Type-2 diabetes mellitus. J. Clin. Invest. 91:
301-307. [0229] 46. Nauck et al. 1993. normalization of fasting
hyperglycemia by exogenous glucagon-like peptide 1 (7-36) amide in
Type 2 (non-insulin-dependent) diabetic patients. Diabetologia. 36:
741-744. [0230] 47. Nishizuka. 1984. The role of protein kinase C
in cell surface signal transduction and tumor promotion. Nature
308: 693-698. [0231] 48. Ochs et al. 1983. Intracellular free
calcium concentrations in isolated pancreatic acini: effects of
secretagogues. Biochem. Biophys. Res. Commun. 117: 122-128. [0232]
49. Offermanns et al. 1993. Stimulation of tyrosine phosphorylation
and mitogen-activated-protein (MAP) kinase activity in human
SH-SY5Y neuroblastoma cells by carbachol. Biochem. J. 294: 545-550.
[0233] 50. Orskov. 1992. Glucagon-like peptide-1, a new hormone of
the entero-insular axis. Diabetologia. 35: 701-711. [0234] 51.
Perfetti et al. 1995. Age-dependent reduction in insulin secretion
and insulin mRNA in isolated islets from rats. Am. J. Physiol. 269:
E983-990. [0235] 52. Physician's Guide to Insulin Dependent [Type
I] Diabetes Mellitus: Diagnosis and Treatment. American Diabetes
Association, 1988. [0236] 53. Rall et al. 1973. Early
differentiation of glucagon-producing cells in embryonic pancreas;
a possible developmental role for glucagon. Proc. Nat. Acad. Sci.
USA 70: 3478-3482. [0237] 54. Ritzel et al. 1995. Pharmacokinetic,
insulinotropic, and glucagonostatic properties of GLP-1 [7-36
amide] after subcutaneous injection in healthy volunteers.
Dose-response-relationships. Diabetologia. 38: 720-725. [0238] 55.
Rivard et al. 1995. Novel model of integration of signaling
pathways in rat pancreatic acinar cells. Am. J. Physiol. 269:
G352-G362. [0239] 56. Saiki et al. 1988. Primer-directed enzymatic
amplification of DNA with a thermostable DNA polymerase. Science
239: 487-491. [0240] 57. Schaffert et al. 1997. Modification of
Blood Group A Expression in Human Pancreatic Tumor Cell Lines by
Inhibitors of N-Glycan Processing. Internat'l J. Pancreatology 21:
21-29. [0241] 58. Schnefel et al. 1990. Cholecystokinin activates
G.sub.i1-, G.sub.i2-, G.sub.i3- and several G.sub.s-proteins in rat
pancreatic acinar cells. Biochem. J. 269: 483-488. [0242] 59.
Siddhanti et al. 1995. Forskolin inhibits protein kinase C-induced
mitogen-activated protein kinase activity in MC3T3-E1 osteoblasts.
Endocrinology. 136:4834-4841. [0243] 60. Spurgeon et al. 1990.
Simultaneous measurement of Ca.sup.2+, contraction, and potential
in cardiac myocytes. Am. J. Physiol. 258: H574-H586. [0244] 61.
Steiner et al. 1972. Radioimmunoassay for cyclic nucleotides II
adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate in
mammalian tissues and body fluids. J. Biol. Chem. 247: 1114-1120.
[0245] 62. Teitelman. Induction of beta-cell neogenesis by islet
injury. Diabetes Metabolism Rev. 12: 91-102, 1996. [0246] 63.
Thorens. 1992. Expression cloning of the pancreatic beta cell
receptor for the gluco-incretin hormone glucagon-like peptide 1.
Proc. Natl. Acad. Sci. USA 89: 8641-8645. [0247] 64. Thorens et al.
1993. Cloning and functional expression of the GLP-1 receptor:
Demonstration that exendin-4 is an agonist and exendin-3(9-39) is
an antagonist of the receptor. Diabetes. 42: 1678-1672. [0248] 65.
Thorens and Waeber. 1993. Glucagon-like peptide-1 and the control
of insulin secretion in the normal state and in NIDDM. Diabetes.
42: 1219-1225. [0249] 66. UK Prospective Study Group. 1995. UK
Prospective Diabetes Study 16: Overview of 6 years' therapy of Type
2 diabetes: A progressive disease. Diabetes. 44: 1249-1258. [0250]
67. Vajanaphanich et al. 1995. Cross-talk between calcium and
cAMP-dependent intracellular signaling pathways. J. Clin. Invest.
96: 386-393. [0251] 68. Valverde and Villanueva-Penacarrillo. 1996.
In vitro insulinomimetic effects of GLP-1 in liver, muscle and fat.
Acta Physiologica Scandinavica 157: 359-360. [0252] 69. von
Weizsacker et al. 1992. Diversity among the beta subunits of
heterotrimeric GTP-binding proteins; Characterization of a novel
beta-subunit cDNA. Biochem. Biophys. Res. Commun. 183: 350-356.
[0253] 70. Wang et al. 1997. Glucagon-like peptides-1 can reverse
the age related decline in glucose tolerance in rats. J. Clin.
Invest. 99: 2883-2889. [0254] 71. Wang et al. 1995. Glucagon-like
peptide-1 affects gene transcription and messenger ribonucleic acid
stability of components of the insulin secretory system in RIN
1046-38 cells. Endocrinology. 136: 4910-4917. [0255] 72. Wang et
al. 1996. GIP regulates glucose transporters, hexokinases, and
glucose-induced insulin secretion in RIN 1046-38 cells. Moll. Cell.
Endo. 116: 81-87. [0256] 73. Wang et al. 1995. Glucagon-like
peptide-1 is a physiological incretin in rat. J. Clin. Invest. 95:
417-421. [0257] 74. Wang et al. 1988. Effects of aging on insulin
synthesis and secretion. Differential effects on proinsulin
messenger mRNA levels, proinsulin biosynthesis, and secretion of
newly made and preformed insulin in the rat. J. Clin. Invest. 81:
176-184. [0258] 75. Wang and Rowe. 1988. Age-related impairment in
the short term regulation of insulin biosynthesis by glucose in rat
pancreatic islets. Endocrinology 123: 1008-1013. [0259] 76. Widmann
et al. 1996. Desensitization and phosphorylation of the
glucagon-like peptide-1 (GLP-1) receptor by GLP-1 and 4-phorbol
12-Myristate 13-acetate. Mol. Endocrinol. 10: 62-75. [0260] 77.
Wills et al. 1996. Gastric emptying, glucose responses, and insulin
secretion after a liquid test meal: effects of exogenous
glucagon-like peptide-1-(7-36) amide in Type 2
(non-insulin-dependent) diabetic patients, J. Clin. Endocrinol.
Metab. 81: 327-332. [0261] 78. Yada et al. Glucagon-like
peptide-1-(7-36) amide and a rise in cyclic adenosine
3',5'-monophosphate increase cytosolic free Ca.sup.2+ in rat
pancreatic .beta.-cells by enhancing Ca.sup.2+ channel activity.
Endocrinology 133: 1685-1692. [0262] 79. Zamponi et al. 1997.
Crosstalk between G proteins and protein kinase C mediated by the
calcium channel alpha 1 subunit. Nature. 385: 442-446. [0263] 80.
Scharp et al. 1991. Transplant. 51: 76. [0264] 81. Warnock et al.
1991. Diabetologia 34: 55.
Sequence CWU 1
1
25 1 30 PRT Human 1 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser
Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu
Val Lys Gly Arg 20 25 30 2 31 PRT Human 2 His Ala Glu Gly Thr Phe
Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys
Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30 3 29 PRT Human
3 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1
5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly 20 25 4
28 PRT Human 4 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr
Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val
Lys 20 25 5 27 PRT Human 5 His Ala Glu Gly Thr Phe Thr Ser Asp Val
Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala
Trp Leu Val 20 25 6 26 PRT Human 6 His Ala Glu Gly Thr Phe Thr Ser
Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe
Ile Ala Trp Leu 20 25 7 25 PRT Human 7 His Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu
Phe Ile Ala Trp 20 25 8 24 PRT Human 8 His Ala Glu Gly Thr Phe Thr
Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu
Phe Ile Ala 20 9 39 PRT Gila monster 9 His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly Ala
Pro Pro Pro Ser 35 10 38 PRT Gila monster 10 His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser
Gly Ala Pro Pro Pro 35 11 37 PRT Gila monster 11 His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30
Ser Gly Ala Pro Pro 35 12 36 PRT Gila monster 12 His Gly Glu Gly
Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala
Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30
Ser Gly Ala Pro 35 13 35 PRT Gila monster 13 His Gly Glu Gly Thr
Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val
Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser
Gly Ala 35 14 34 PRT Gila monster 14 His Gly Glu Gly Thr Phe Thr
Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu
Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 25 30 Ser Gly 15 33
PRT Gila monster 15 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys
Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu
Lys Asn Gly Gly Pro Ser 20 25 30 Ser 16 32 PRT Gila monster 16 His
Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 10
15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser
20 25 30 17 31 PRT Gila monster 17 His Gly Glu Gly Thr Phe Thr Ser
Asp Leu Ser Lys Gln Met Glu Glu 1 5 10 15 Glu Ala Val Arg Leu Phe
Ile Glu Trp Leu Lys Asn Gly Gly Pro 20 25 30 18 30 PRT Gila monster
18 His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly
20 25 30 19 38 DNA Artificial Sequence Oligonucleotide primer 19
gatggatcct gcagaagctt tttttttttt tttttttt 38 20 19 DNA Artificial
Sequence Oligonucleotide primer 20 acaggtctct tctgcaacc 19 21 20
DNA Artificial Sequence Oligonucleotide primer 21 aagatgactt
catgcgtgcc 20 22 28 DNA Artificial Sequence Oligonucleotide primer
22 tgcccaggct tttgtcaaac agcacctt 28 23 20 DNA Artificial Sequence
Oligonucleotide primer 23 ctccagtgcc aaggtctgaa 20 24 27 DNA
Artificial Sequence Oligonucleotide primer 24 gtggctggat tgtttgtaat
gctgctg 27 25 24 DNA Artificial Sequence Oligonucleotide primer 25
cggttcctct tggtgttcat caac 24
* * * * *