Methods of treating non-insulin dependent diabetes mellitus with pancreatic polypeptide

The present invention provides a method of treating NIDDM in a patient diagnosed with NIDDM by administering to the patient a compound in a pharmaceutically acceptable carrier that reduces hepatic glucose production in the patient by inhibiting hepatic expression of the alpha subunit of a G.sub.s protein in a liver cell plasma membrane, thereby inhibiting stimulation of cAMP by glucagon, whereby the reduction in hepatic glucose production treats the NIDDM. Also provided is a method for screening compounds for the ability to treat NIDDM comprising determining if the compound decreases hepatic expression of the alpha subunit of a G.sub.s protein in a liver cell plasma membrane, thereby inhibiting the stimulation of cAMP by glucagon, a compound which decreases the hepatic expression of the alpha subunit of the G.sub.s protein in the liver cell plasma membrane, thereby inhibiting the stimulation of cAMP by glucagon, being a compound with the ability to treat NIDDM. The present invention further provides a kit for treating NIDDM comprising a compound in a pharmaceutically acceptable carrier that decreases hepatic expression of the alpha subunit of the G.sub.s protein in the liver cell plasma membrane, thereby inhibiting stimulation of cAMP by glucagon.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a method for treating non-insulin 
dependent diabetes mellitus (NIDDM) in a human subject. In particular, the 
present invention relates to the administration of pancreatic polypeptide 
or the carboxyl terminal fragment of pancreatic polypeptide, either alone 
or in combination with insulin or an oral hypoglycemic agent to treat 
NIDDM by a) inhibiting stimulation of cyclic adenosine monophosphate 
(cAMP) by glucagon, b) inhibiting secretion of digestive enzymes from the 
exocrine pancreas, and/or c) potentiating the effect of leptin in reducing 
neuropeptide Y synthesis. 
2. Background Art 
NIDDM, also known as type II diabetes or maturity onset diabetes (MOD) 
describes a disorder, primarily in adults, characterized by fasting or 
post-meal hyperglycemia and most commonly associated with obesity. NIDDM 
is distinguished from type I diabetes or insulin dependent diabetes on the 
basis that the type II diabetic is not dependent on insulin for survival. 
Most patients diagnosed with NIDDM exhibit elevated basal insulin levels, 
with the degree of elevation often correlated with the degree of obesity. 
Although NIDDM is correlated with resistance to insulin action, the primary 
defect that induces this disease has not yet been established. The 
metabolic dysfunctions associated with NIDDM are insulin resistance 
(impaired insulin sensitivity) and increased hepatic glucose output 
(hyperglycemia). Insulin resistance describes a patho-physiological state 
in which insulin does not produce the expected decrease in blood glucose 
concentrations. Insulin sensitivity refers to a complete or partial 
reversal of the insulin resistant state. 
Most current therapies prescribed for treatment of NIDDM act by stimulating 
insulin release, which may actually be detrimental because this leads to 
early exhaustion of the pancreatic islets. Furthermore, high insulin 
levels may contribute to complications of the disease. Thus, there exists 
a need for a treatment of NIDDM that results in a decrease in 
hyperglycemia and an increase in insulin sensitivity without additional 
detrimental effects. 
The present invention fulfills this need by providing an effective 
treatment of NIDDM by the administration of an amount of pancreatic 
polypeptide (PP) alone or in combination with other hypoglycemic agents. 
SUMMARY OF THE INVENTION 
The present invention provides a method of treating NIDDM in a patient 
diagnosed with NIDDM by administering to the patient a compound, in a 
pharmaceutically acceptable carrier, that reduces hepatic glucose 
production in the patient by inhibiting hepatic expression of the alpha 
subunit of a G.sub.s protein in a liver cell plasma membrane, thereby 
inhibiting stimulation of cAMP by glucagon, whereby the reduction in 
hepatic glucose production treats the NIDDM. 
Also provided is a method for screening compounds for the ability to treat 
NIDDM comprising determining if the compound decreases hepatic expression 
of the alpha subunit of a G.sub.s protein in a liver cell plasma membrane, 
thereby inhibiting the stimulation of cAMP by glucagon, a compound which 
decreases the hepatic expression of the alpha subunit of the G.sub.s 
protein in the liver cell plasma membrane, thereby inhibiting the 
stimulation of cAMP by glucagon, being a compound with the ability to 
treat NIDDM. 
The present invention further provides a kit for treating NIDDM comprising 
a compound in a pharmaceutically acceptable carrier that decreases hepatic 
expression of the alpha subunit of the G.sub.s protein in the liver cell 
plasma membrane, thereby inhibiting stimulation of cAMP by glucagon. 
Also provided is a method for treating NIDDM in a patient diagnosed with 
NIDDM by administering to the patient a compound, in a pharmaceutically 
acceptable carrier, that inhibits beta cell and pancreatic cell 
hypertrophy by binding the vagal nuclear complex, thereby inhibiting 
secretion of digestive enzymes by the exocrine pancreas in the patient, 
whereby the inhibition of beta cell and pancreatic islet hypertrophy 
treats the NIDDM. 
In addition, the present invention provides a method of screening compounds 
for the ability to treat NIDDM comprising determining if the compound 
binds the vagal nuclear complex and inhibits secretion of digestive 
enzymes by the exocrine pancreas, thereby inhibiting beta cell and 
pancreatic islet hypertrophy, a compound which binds the vagal nuclear 
complex and inhibits secretion of digestive enzymes by the exocrine 
pancreas, thereby inhibiting beta cell and pancreatic islet hypertrophy, 
being a compound with the ability to treat NIDDM. 
Also provided is a kit for treating NIDDM comprising a compound in a 
pharmaceutically acceptable carrier that binds the vagal nuclear complex 
and inhibits secretion of digestive enzymes by the exocrine pancreas, 
thereby inhibiting beta cell and pancreatic islet hypertrophy. 
The present invention further provides a method for treating NIDDM in a 
patient diagnosed with NIDDM by administering to the patient a compound, 
in a pharmaceutically acceptable carrier, that enhances insulin 
sensitivity and reverses the effects of neuropeptide Y by binding the 
arcuate nucleus in the hypothalamus, thereby potentiating the effect of 
leptin in reducing neuropeptide Y synthesis, whereby the enhancement of 
insulin sensitivity and reversal of the effects of neuropeptide Y treat 
the NIDDM. 
Additionally provided is a method of screening compounds for the ability to 
treat NIDDM comprising determining if the compound binds the arcuate 
nucleus in the hypothalamus and potentiates the effect of leptin in 
reducing neuropeptide Y synthesis, thereby enhancing insulin sensitivity 
and reversing the effects of neuropeptide Y, a compound which enhances 
insulin sensitivity and reverses the effects of neuropeptide Y being a 
compound with the ability to treat NIDDM. 
Furthermore, the present invention provides a kit for treating NIDDM 
comprising a compound in a pharmaceutically acceptable carrier that binds 
the arcuate nucleus in the hypothalamus and potentiates the effect of 
leptin in reducing neuropeptide Y synthesis, thereby enhancing insulin 
sensitivity and reversing the effects of neuropeptide Y. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention may be understood more readily by reference to the 
following detailed description of specific embodiments and the Examples 
included herein. As used herein, "a" can included multiples. 
This invention provides a method of treating NIDDM in a patient diagnosed 
with NIDDM by administering to the patient a compound, in a 
pharmaceutically acceptable carrier, that reduces hepatic glucose 
production in the patient by decreasing hepatic expression of the alpha 
subunit of a G.sub.s protein in a liver cell plasma membrane, thereby 
inhibiting stimulation of cAMP by glucagon in the patient, whereby the 
reduction in hepatic glucose production treats the NIDDM. 
The primary action of glucagon (circulating hormone from pancreas) in the 
liver is to promote glycogenolysis (breakdown of glycogen to produce 
glucose) and it produces this effect by binding to its cell surface 
receptor, activating the heterotrimeric stimulatory G protein (G.sub.s) 
and promoting its dissociation into a monomeric .alpha. subunit and 
dimeric .beta..gamma. subunits. The activated G.sub.s .alpha. subunit 
interacts with and activates adenylyl cyclase, which increases the 
conversion of ATP to cyclic AMP. The increase in cellular cyclic AMP 
activates cAMP-dependent protein kinase, which phosphorylates and 
activates phosphorylase kinase. Once activated, this enzyme phosphorylates 
and activates glycogen phosphorylase, with a net result being increased 
breakdown of glycogen into glucose-1-phosphate. In NIDDM, where insulin 
resistance compromises the normal ability of insulin to stimulate 
peripheral glucose uptake, glucagon's glycogenolytic effects exacerbate 
the existing hyperglycemia. A decrease in the ability of glucagon to 
stimulate cyclic AMP production results in a decrease in hepatic glucose 
production and has the net effect of lessening the severity of 
hyperglycemia associated with NIDDM. 
The compound of this invention can be PP, having the amino acid sequence: 
Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asn-Ala-Thr-Pro-Glu-Gln-Met-Ala-Gl 
n-Tyr-Ala-Ala-Asp-Leu-Arg-Arg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-NH.su 
b.2 (SEQ ID NO: 1), or the carboxyl terminal fragment of PP, having the 
amino acid sequence: Leu-Thr-Arg-Pro-Arg-Tyr-NH.sub.2 (SEQ ID NO:2). 
Either of these compounds can be administered alone or in combination with 
insulin or other oral hypoglycemic compounds which enhance the function of 
either PP or the carboxyl terminal fragment of PP in reducing hepatic 
glucose production. Alternatively, the compound of the present invention 
can be any compound or combination of compounds that reduce hepatic 
glucose production, in a patient diagnosed with NIDDM, by inhibiting 
expression of the alpha subunit of the G.sub.s protein in the liver cell 
plasma membrane, thereby inhibiting stimulation of cAMP by glucagon in a 
patient diagnosed with NIDDM. 
The present invention further provides a method for treating NIDDM in a 
patient diagnosed with NIDDM by administering to the patient a compound, 
in a pharmaceutically acceptable carrier, that inhibits beta cell and 
pancreatic islet hypertrophy in a patient by binding the vagal nuclear 
complex, thereby inhibiting secretion of digestive enzymes by the exocrine 
pancreas in the patient, whereby the inhibition of beta cell and 
pancreatic islet hypertrophy treats the NIDDM. 
The ordered arrangement of the endocrine cell types within the pancreatic 
islet appears to be an important prerequisite for the normal insulin 
response to a meal. The beta cells which synthesize and secrete insulin 
occur as a central core in the islet and are surrounded by the other islet 
cell types-glucagon cell, pancreatic polypeptide cell and the somatostatin 
cell. The pancreatic islet and beta cell hypertrophy that occurs in 
maturity onset diabetes is associated with a disturbance in this ordered 
architecture and abnormal insulin secretion. Vagal hyperactivity leads to 
islet cell and beta cell hypertrophy and the early (cephalic-vagal) phase 
of insulin secretion is abnormal in NIDDM. It is believed that enhanced 
vagal tone leads to islet and beta cell hypertrophy and disordered insulin 
release. PP inhibits the secretion of pancreatic digestive enzymes by 
inhibiting vagal tone. Furthermore, circulating PP binds to specific 
receptors in the vagal nuclear complex. The vagal nuclear complex (VNC) 
lies in the midbrain and receives incoming information from the vagal 
nerves through the Nucleus Tractus Solitarius. The Dorsal Motor Nucleus of 
the vagus which is also a component of the VNC provides the "efferent" 
nerve fibers that stimulate early phase insulin release and digestive 
enzyme secretion. Furthermore, direct injection of PP into the VNC has 
been shown to inhibit secretion of pancreatic digestive enzymes. In whole 
animals, PP inhibits insulin release. It appears that PP inhibits islet 
cell hypertrophy and insulin release by a direct action on the VNC that 
results in decreased vagal tone to both the pancreatic islet that produces 
insulin and the pancreatic acinar tissue that produces the pancreatic 
digestive enzymes. 
In this embodiment, either PP or the carboxy terminal fragment of PP can be 
administered alone or in combination with insulin or other oral 
hypoglycemic compounds which enhance the function of either PP or the 
carboxyl terminal fragment of PP in inhibiting beta cell and pancreatic 
islet hypertrophy. Alternatively, the compound of the present invention 
can be any compound or combination of compounds that inhibit beta cell and 
pancreatic islet hypertrophy in a patient diagnosed with NIDDM by binding 
the vagal nuclear complex and inhibiting secretion of digestive enzymes by 
the exocrine pancreas in a patient diagnosed with NIDDM. 
Additionally provided in the present invention is a method for treating 
NIDDM in a patient diagnosed with NIDDM by administering to the patient a 
compound, in a pharmaceutically acceptable carrier, that enhances insulin 
sensitivity and reverses the effect of neuropeptide Y (NPY) in a patient 
by binding the arcuate nucleus in the hypothalamus, thereby potentiating 
the effect of leptin in reducing NPY synthesis in the patient, whereby the 
enhancement of insulin sensitivity and reversal of the effects of NPY 
treat the NIDDM. 
NPY is a neurotransmitter that is structurally related to pancreatic 
polypeptide. When injected directly into the brain, it is the most potent 
stimulant to trigger eating known. Indeed it is the only peptide that, 
when injected repeatedly into the brain, causes hyperphagia and obesity. 
NPY also acts through the VNC and vagus nerve to stimulate cephalic phase 
insulin release. Leptin is a peptide released by fat cells which inhibits 
NPY synthesis by an effect on the arcuate nucleus in the hypothalamus. PP 
likely also inhibits NPY synthesis via an action on the PP receptors 
recently identified in the arcuate nucleus. As such it would inhibit the 
ability of NPY to initiate cephalic phase and stimulate early phase 
insulin release by both inhibiting NPY synthesis and decreasing the 
stimulatory effects of NPY on the VNC. 
Current data suggest that increased Neuropeptide Y (NPY) synthesis in the 
arcuate nucleus in the brain causes enhanced food intake, increased vagal 
tone and decreased sympathetic tone; these are features that characterize 
obesity and diabetes. High vagal tone leads to inappropriate insulin and 
glucagon release which coupled with hyperphagia contributes to insulin 
resistance, obesity and diabetes. The data suggest that Pancreatic 
Polypeptide (PP) reverses this syndrome by decreasing vagal tone, 
decreasing insulin release and hepatic glucose production, thereby 
enhancing insulin sensitivity. Insulin stimulates leptin release from 
isolated adipocytes. Thus, PP is expected to inhibit leptin release in the 
whole animal. Furthermore, PP treatment is expected to inhibit NPY mRNA 
expression in the arcuate nucleus based on the demonstration of a novel PP 
receptor population in the hypothalamus using receptor autoradiography. 
Receptor localization in the arcuate nucleus using these techniques 
provides a mechanism to identify hormones and neurotransmitters that would 
be expected to alter food intake and nutrient metabolism by effects on 
NPY. NPY message and peptide levels can be measured directly using methods 
described in the references on the attached sheet. Although much attention 
has been focused recently on leptin, leptin therapy of obesity may not be 
beneficial because of leptin resistance. Given PP's lack of side effects 
even in supra-maximal doses, PP has the potential to be a more viable and 
safe method of treating the obese, diabetic patient. PP appears to 
complete a series of negative feedback loops that serves to control body 
weight and glucose metabolism by modulating insulin release acutely and 
NPY synthesis chronically. These PP feedback loops are impaired in 
diabetics because of decreased secretion rather than down regulation of 
the receptor or impaired signal transduction. First PP can act through 
receptors recently identified in the arcuate nucleus to inhibit NPY 
synthesis. This would lead to decreased food intake, particularly 
decreased intake of carbohydrate rich foods. In addition NPY increases 
vagal tone acting through neural tracts that pass through the 
paraventricular nucleus (PVN) to the Vagal Nuclear Complex (VNC) in the 
brainstem. There are PP receptors in the PVN and the VNC which provides 
multiple sites for PP to inhibit vagal tone. Indeed, PP injected directly 
into the Dorsal Motor Nucleus of the vagus (a component of the VNC) does 
inhibit the vagus. Inhibition of vagal tone to the pancreatic islets would 
explain PP's ability to inhibit insulin release. When this is coupled to 
inhibition of hepatic glucose production (mediated by the PP receptors in 
the liver) insulin sensitivity is enhanced and an improvement in the 
diabetes is observed. PP treatment would have the added benefit that it 
would also decrease body weight. As obesity frequently accompanies 
maturity onset (Type II) diabetes a double benefit would be experienced by 
these patients. 
In this embodiment, either PP or the carboxy terminal fragment of PP can be 
administered alone or in combination with insulin or other oral 
hypoglycemic compounds which enhance the function of either PP or the 
carboxyl terminal fragment of PP in enhancing insulin sensitivity and 
reversing the effects of neuropeptide Y. Alternatively, the compound of 
the present invention can be any compound or combination of compounds that 
enhances insulin sensitivity and reverses the effects of neuropeptide Y in 
a patient diagnosed with NIDDM by binding the arcuate nucleus in the 
hypothalamus and potentiating the effect of leptin in reducing 
neuropeptide Y synthesis in a patient diagnosed with NIDDM. "Syndrome X" 
describes a constellation of abnormal lipid profile, atherosclerosis and 
heart disease associated with and possibly caused by the insulin resistant 
state. PP treatment would also be expected to be beneficial in "Syndrome 
X" and other insulin resistant states. Complications of NIDDM such as 
renal failure, diabetic retinopathy, atherosclerosis, cardiovascular 
disease and neuropathy could be delayed or made less severe by treatment 
with PP. PP reverses the insulin resistant state, thus establishing a 
state of insulin sensitivity. 
The PP and carboxyl terminal fragment of PP can be obtained by standard 
procedures known in the art and are also available from commercial sources 
(e.g., Peninsula Labs, Belmont, Calif.). Recombinant human insulin can be 
obtained either commercially (e.g. Eli Lilly, Indianapolis, Ind.), or by 
standard procedures known in the art. 
The PP can be administered in a range between 2 and 500 .mu.g/kg of body 
weight/day, preferably in a range between 4 and 100 .mu.g/kg of body 
weight/day and most preferably in a dosage of 12 .mu.g/kg body weight/day. 
Chemical alteration of the peptide, as described below, could lead to 
longer acting analogs (e.g. substitution of D-amino acids, etc.), 
requiring even lower dosages such as for example, between 0.5 and 50 
.mu.g/kg of body weight/day. This amount can be administered as a single 
dose or divided into several doses to be administered over a 24 hour 
period. The exact dosage may vary on the basis of the patient's age, 
weight, size and general overall condition and a physician would best be 
able to determine the exact dosage according to these parameters. Further 
guidance on determining dosages and modes of administration are available 
as provided in Remington's Pharmaceutical Sciences (13). 
Analogs of PP and of the carboxyl terminal fragment of PP can also be 
administered to patients to treat NIDDM. Such analogs can include, but are 
not limited to, compounds having the chemical composition of PP or the 
carboxyl terminal fragment of PP, wherein D-amino acids have been 
incorporated into the molecule. Such analogs would be anticipated to have 
a longer acting effect in humans than PP and the carboxyl terminal 
fragment of PP and would, thus, be administered in lower doses. For 
example, a substitution of a D-amino acid at the amino terminal of the 
carboxyl terminal fragment of PP would protect the peptide from enzymatic 
degradation: 
D-Leu-Thr-Arg-Pro-Arg-Tyr-NH2 (SEQ ID NO:3). Other examples of D-amino acid 
substitutions than can be beneficial are: 
D-Leu-Thr-Arg-Pro-D-Arg-Tyr-NH.sub.2 (SEQ ID NO:4) and 
Leu-Thr-Arg-Pro-D-Arg-Tyr-NH.sub.2 (SEQ ID NO:5). 
The carboxyl terminal fragment of PP can be administered to a patient 
diagnosed with NIDDM in a range between 20 and 5,000 .mu.g/kg of body 
weight/day, more preferably in a range of 40 to 1,000 .mu.g/kg of body 
weight/day and most preferably in a dosage of 200 .mu.g/kg of body 
weight/day. Chemical alteration of the fragment of PP could lead to longer 
acting analogs (e.g. substitution of D-amino acids, etc.), requiring even 
lower dosages such as for example, between 1 and 1,000 .mu.g/kg of body 
weight/day. This amount can be administered as a single dose or divided 
into several doses to be administered over a 24 hour period. The exact 
dosage may vary on the basis of the patient's age, weight, size and 
general overall condition and a physician would best be able to determine 
the exact dosage according to these parameters. Further guidance on 
determining dosages and modes of administration are available as provided 
in Remington's Pharmaceutical Sciences (13). 
PP and carboxyl terminal fragments and analogs could be administered with 
injectable insulin or with other hypoglycemic agents. These include, but 
are not limited to, oral hypoglycemic compounds such as the sulfonylureas 
and the biquanides. The sulfonylureas can include, but are not limited to, 
Tobutanine (Orinase) Acetohexamide (Dymelor), Tolazanide (Tolinase) and 
Chloropropramide (Diabenase). Biquanides can include, but are not limited 
to, glyburide, glopizide and metformin. 
The compounds administered to treat NIDDM can be administered orally and/or 
parenterally in a pharmaceutically acceptable carrier to human subjects. 
The preferable mode of administration would be by subcutaneous injection. 
Transnasal and transdermal administrations or any other oral or parenteral 
route are other potential modes of administration. Suitable carriers for 
use in the present invention include, but are not limited to, pyrogen-free 
saline. For parenteral administration of the compounds, a sterile solution 
or suspension is prepared in saline that may contain additives, such as 
ethyl oleate or isopropyl myristate, and can be injected, for example, 
into subcutaneous or intramuscular tissues. 
Suitable carriers for oral administration of compounds to treat NIDDM can 
include one or more substances which may also act as flavoring agents, 
lubricants, suspending agents, or as protectants. Suitable solid carriers 
include calcium phosphate, calcium carbonate, magnesium stearate, sugars, 
starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans. 
Suitable liquid carriers may be water, pharmaceutically accepted oils, or 
a mixture of both. The liquid can also contain other suitable 
pharmaceutical additions such as buffers, preservatives, flavoring agents, 
viscosity or osmo-regulators, stabilizers or suspending agents. Examples 
of suitable liquid carriers include water with or without various 
additives, including carboxypolymethylene as a pH-regulated gel. The 
compounds may be contained in enteric coated capsules that release the 
compounds into the intestine to avoid gastric breakdown. 
Alternatively, the compounds may be microencapsulated with either a natural 
or a synthetic polymer into microparticles 4-8 .mu.m in diameter, which 
target intestinal lymphoid tissues and produce a sustained release of 
compounds for up to four weeks (14, 15). 
This invention further provides a method of screening substances for the 
ability to treat NIDDM, comprising determining if the compound is an 
inhibitor of the expression of the alpha subunit of the G.sub.s proteins 
in the liver cell plasma membrane, thereby inhibiting the stimulation of 
cAMP by glucagon, a compound which inhibits the expression of the alpha 
subunit of the G.sub.s proteins in the liver cell plasma membrane, thereby 
inhibiting the stimulation of cAMP by glucagon, being a compound with the 
ability to treat NIDDM. For example, the compound can be administered to 
an appropriate animal model as described below and the efficacy of the 
compound in inhibiting the ability of glucagon to activate cyclic AMP can 
be determined according to the experimental protocols set forth in the 
Examples herein. 
The present invention further provides a method of screening compounds for 
the ability to treat NIDDM, comprising determining if the compound binds 
the vagal nuclear complex and inhibits secretion of digestive enzymes by 
the exocrine pancreas, thereby inhibiting beta cell and pancreatic islet 
hypertrophy. A compound which binds the vagal nuclear complex and inhibits 
secretion of digestive enzymes by the exocrine pancreas inhibits beta cell 
and pancreatic islet hypertrophy is a compound with the ability to treat 
NIDDM. For example, the compound can be administered to an appropriate 
animal model as described below and the efficacy of the compound in 
binding the vagal nuclear complex and inhibiting secretion of digestive 
enzymes by the exocrine pancreas can be determined according to the 
experimental protocols set forth in the Examples herein. Assays for 
determining the ability of a compound to bind the vagal nuclear complex 
are known in the art. 
Also provided in the present invention is a method of screening compounds 
for the ability to treat NIDDM, comprising determining if the compound 
binds the arcuate nucleus in the hypothalamus and potentiates the effect 
of leptin in reducing NPY synthesis, thereby enhancing insulin sensitivity 
and reversing the effects of NPY. A compound which enhances insulin 
sensitivity and reverses the effects of NPY can be used to treat NIDDM. 
For example, the compound can be administered to an appropriate animal 
model as described below and the efficacy of the compound in binding the 
arcuate nucleus in the hypothalamus and potentiating the effect of leptin 
in reducing NPY synthesis can be determined according to the experimental 
protocols set forth in the Examples herein. Assays that can be used to 
determining the ability of PP and other compounds to effect NPY synthesis 
in the arcuate nucleus and PVN are known in the art. For example, methods 
that could be used to measure NPY levels in specific regions of the brain 
after PP treatment (arcuate nucleus and PVN) are included in references 
23-29. Methods that could be used to measure NPY message levels in 
specific regions of the brain are also included in references (25-27). 
The preferred animal models that could be used for screening for the 
efficacy of various compounds to treat NIDDM include but are not limited 
to: 1) the fatty Zucker (fa/fa) rat, 2) the ob/ob mouse, 3) the db/db 
mouse, as well as any other suitable animal model of hyperglycemia, 
obesity and impaired insulin function now known or developed in the 
future. These models are produced by recessive inheritance of a single 
gene (1,5,8,9). Other models can include 1) a diet-induced obesity rodent 
model, wherein specific strains of rodents are fed a high fat or 
"cafeteria" type diet, 2) a VMH-lesioned animal model and an animal model 
wherein repeated injections of NPY are given by indwelling cannula into 
the cerebroventricular system of the brain, and 3) humans diagnosed with 
NIDDM. 
In another embodiment, the present invention provides a kit for treating 
NIDDM comprising a compound in a pharmaceutically acceptable carrier that 
inhibits the expression of the alpha subunit of the G.sub.s proteins in 
the liver cell plasma membrane, thereby inhibiting the stimulation of cAMP 
by glucagon. 
A kit for treating NIDDM is also provided, comprising a compound in a 
pharmaceutically acceptable carrier that binds the vagal nuclear complex 
and inhibits secretion of digestive enzymes by the exocrine pancreas, and 
beta cell and pancreatic islet hypertrophy. 
Additionally, the present invention provides a kit for treating NIDDM 
comprising a compound in a pharmaceutically acceptable carrier that binds 
the arcuate nucleus in the hypothalamus and potentiates the effect of 
leptin in reducing neuropeptide Y synthesis, thereby enhancing insulin 
sensitivity and reversing the effects of neuropeptide Y. 
The compound of the kit can be PP or the carboxyl terminal fragment of PP, 
either alone or in combination with insulin or other oral-hypoglycemic 
agents. Alternatively, the compound of the kit can be a compound 
determined to have the ability to treat NIDDM, according to the protocols 
taught in the Examples herein. 
The following Examples are intended to illustrate, but not limit, the 
invention. While the protocols described are typical of those that might 
be used, other procedures known to those skilled in the art may be 
alternatively employed.

EXAMPLES 
Experimental Animal Protocol. Lean (+/?) and fatty (fa/fa) Zucker rats were 
obtained from Harlan Labs at seven weeks of age and randomly assigned to 
two groups within each phenotype. One group was injected subcutaneously 
with PP (Peninsula Labs (Belmont, Calif.) (200 .mu.g/day/kg body weight) 
in a vehicle of physiological saline (0.9% w/v) while the other group was 
injected with only vehicle. The two groups within each phenotype received 
their respective treatments for five days, after which the animals were 
weighed and sacrificed following an overnight fast. Blood samples were 
obtained from each animal for assays of plasma glucose and insulin 
according to standard methods known in the art (3) and the pancreas was 
removed and subjected to an acid-ethanol extraction (0.1N HCl, 70% 
ethanol) as described previously for assay of pancreatic insulin (3). 
Livers were removed into ice cold balanced salt solution. After cooling, 
each liver was cleaned of connective tissue and minced into 2-4 mm pieces 
with scissors in 5 ml of ice-cold homogenizing buffer (50 mM Hepes, pH 
6.5, 5 mM EDTA, 3 mM orthophenanthroline, 0.2 .mu.M iodoacetic acid, 1 mM 
PMSF, 25 .mu.m leupeptin (3). The minced liver was homogenized in a 40 ml 
Dounce homogenizer in 25 ml of homogenizing buffer with 25 strokes of the 
loose pestle and three strokes of the tight pestle. The homogenate was 
centrifuged for five minutes at 3000.times.g. The supernatant was removed 
and centrifuged for 20 min at 16,000.times.g. The resulting pellet was 
retained, resuspended in phosphate buffered saline (PBS) containing 5 mM 
MgCl.sub.2 and homogenized with five strokes of the Dounce homogenizer. 
After centrifuging for 20 min at 16,000.times.g, the membrane pellet was 
resuspended in homogenizing buffer containing 0.1% bovine serum albumin 
(BSA). Aliquots were frozen in liquid nitrogen and stored at -80.degree. 
C. 
Preparation of PP and the carboxyl terminus of PP. Highly purified PP and 
the C-terminal hexapeptide of PP were obtained from commercial sources. 
Glucagon binding in liver membranes. Glucagon was radioiodinated using 
chloramine T and purified by C-18 reverse phase HPLC. In brief, 25 .mu.g 
of glucagon was incubated with 1 mCi of carrier free Na.sup.125 I in the 
presence of 1 .mu.g chloramine T for 30 seconds. The reaction was 
terminated with 2 .mu.g sodium metabisulfite, followed by purification of 
monoiodinated glucagon by HPLC. The labeled glucagon was diluted with an 
equal volume of 25 mM Tris buffer (pH 7.4) containing 1% BSA and stored at 
4.degree. C. prior to use. The glucagon receptor binding assay was 
conducted by a modification of the procedure of Lin et al. (12) in a final 
volume of 500 .mu.l. Saturation binding experiments were conducted with 25 
.mu.g of liver plasma membranes and 10-5000 pM radiolabeled glucagon. The 
samples were incubated for one hour at 30.degree. C., and membrane bound 
glucagon was isolated by centrifugation at 40,000.times.g for ten minutes. 
Non-specific binding was assayed at each ligand concentration by including 
1 .mu.M unlabeled glucagon and was determined to average 10-20%. 
Competition binding curves were conducted using 50 pM labeled glucagon. 
Binding curves were analyzed using nonlinear least squares. 
Saturation binding studies provided no evidence that the total number of 
glucagon binding sites differed between phenotype (lean control B.sub.max 
=475.+-.23 fmol/mg; fatty control B.sub.max =471.+-.44 fmol/mg). In 
addition, treatment with PP failed to alter total glucagon binding sites 
in either phenotype (fatty PP B.sub.max 569.+-.45 fmol/mg). The confidence 
intervals for the means in question are: 475.+-.23 fmol/mg, 95% CI (420.1 
to 529.9 fmol/mg) lean control group; 471.+-.44 fmol/mg, 95% CI (366.2 to 
576.0 fmol/mg) fatty control group; and 569.+-.45 fmol/mg, 95% CI (462.3 
to 676.8 fmol/mg) fatty PP group. The same conclusion was reached after 
analysis of binding data from a second experiment. The data from both 
experiments indicate that the differences in efficacy of glucagon in 
activating adenylyl cyclase to form cAMP between the phenotypes and its 
modulation by PP treatment are probably not the result of differences in 
glucagon receptor expression. Notwithstanding these similarities, the 
Scatchard plots suggest that binding affinities for glucagon may differ 
among the groups. However, comparison of estimates of K.sub.d among the 
groups (lean control: 0.39.+-.0.16 nM; fatty control: 0.85.+-.0.19 nM; 
fatty PP: 1.70.+-.0.25 nM) produced wide confidence intervals and 
precluded detection of treatment differences. 
A more rigorous test for group differences in binding affinity was provided 
by competition binding experiments with 50 pM labeled glucagon in the 
presence and absence of GTP. The K.sub.d values estimated using this 
method did not differ among the groups (lean control: 0.49.+-.0.06 nM; 
fatty control: 0.86.+-.0.09 nM; fatty PP: 0.66.+-.0.09 nM). This approach 
also provides estimates of the proportion of receptors existing in high 
versus low affinity states and revealed that the percentage of high 
affinity binding sites in the fatty control group was elevated (68%) 
compared to the lean control group (53%). This phenotypic difference was 
not altered by treatment with PP (lean PP: 55% high affinity; fatty PP: 
71% high affinity). These data indicate that a decrease in the total 
number of glucagon receptors is not the mechanism of PP's effects on the 
efficacy of glucagon to activate cyclic AMP. 
Receptor dependent labeling of hepatocyte G proteins with 
4-Azidoanilido-.alpha..sup.32 P!GTP. The protocol for labeling G proteins 
with AA-GTP was as described previously (11). The AA-32P!-GTP used in the 
labeling experiments was prepared according to the protocol of Offermans 
et al. (6,7). 
The purpose of these labeling studies was to determine whether PP 
stimulated the binding of AA-.sup.32 P!GTP to proteins migrating at the 
known molecular weight of G proteins in liver plasma membranes. 
Angiotensin II was used as a positive control because it has been shown to 
couple to pertussis-sensitive G proteins (16). Incubation of liver plasma 
membranes with 1 .mu.M angiotensin II produced a two-fold increase in 
labeling of G protein(s) migrating at 41 kDa on SDS PAGE gels The 41 kDa 
band represents the pertussis-sensitive G protein family that is expressed 
in hepatocytes and which couples to angiotensin II receptors. 
Experiments of comparable design using PP instead of angiotensin II 
illustrated that PP also produces a concentration-dependent increase in 
labeling of G proteins migrating at 41 kDa. The maximal increase in 
labeling was approximately two-fold and was comparable to the maximal 
effect seen with angiotensin II. The data describing PP dependent labeling 
of liver G proteins with AA-.sup.32 P!GTP illustrate early steps in the 
PP signaling cascade. The intervening steps between PP dependent G protein 
activation and decreased efficacy of glucagon have not been determined. 
Adenylyl Cyclase Assay. Adenylyl cyclase activity leading to the production 
of cAMP was determined in liver plasma membranes by methods previously 
described (2, 4). Briefly, 25 .mu.g of liver plasma membranes were 
incubated for ten minutes at 30.degree. C. in a buffer containing 50 mM 
TES (pH 7.4), 4.0 mM MgCl.sub.2, 2 mM creatine phosphate, 25 U/ml creatine 
phosphokinase, 100 .mu.M ATP and 10 .mu.M GTP. The reaction was conducted 
in a final volume of 300 .mu.l and initiated by adding 50 .mu.l of the 
membrane preparation to each incubation tube. Reactions were terminated by 
adding 50 .mu.l of cold 25% TCA and centrifuging for 15 min at 3000 rpm. 
Cyclic AMP formed in the reaction was measured in the supernatant by 
radioimmunoassay according to methods described previously (2, 4). 
Dose-response curves were characterized using the four parameter logistic 
ogive in relation to log dose as described previously (2, 4). 
Glucagon produced a concentration-dependent activation of adenylyl cyclase 
in liver plasma membranes from all four experimental groups. The response 
curve for the lean group treated with PP was similar to the control lean 
group. The estimated potencies did not differ among the groups and ranged 
from 5-15 nM in five experimental replicates. Maximal adenylyl cyclase 
activation by glucagon was much higher in fatty than in lean Zucker rats 
(58.1.+-.4.4 vs. 36.4.+-.1.8 pmol cAMP/min/mg membrane protein). 
Treatment of lean rats with PP did not modify the efficacy of glucagon in 
liver membranes. In contrast, treatment of fatty Zucker rats with PP as 
described above for five days produced a significant decrease in the 
efficacy of glucagon in activating adenylyl cyclase, reducing the maximal 
adenylyl cyclase activation to levels not different from lean Zucker rats. 
A second replicate of this experiment with additional animals produced a 
similar decrease in maximal adenylyl cyclase activation in PP treated 
fatty Zucker rats (60.2.+-.1.5 pmol cAMP/min/mg) compared to 
vehicle-treated fatty Zucker rats (87.6.+-.1.8 pmol cAMP/min/mg). These 
results indicate that livers from fatty Zucker rats are hyper-responsive 
to glucagon compared to their lean litter mates. These studies further 
demonstrate that chronic treatment of fatty Zucker rats with PP (i.e., 
treatment for five days), as described above, corrects the 
hyper-responsiveness of fatty Zucker rats to glucagon. 
Western Blot assay of G.sub.s .alpha.. Antibodies were generated against 
the C-terminal decapeptide (aa 345-354) of G.sub.s .alpha. conjugated to 
keyhole limpet cyanin (KLH) via a cysteine placed on the N-terminal end of 
each peptide (18). Rabbits were immunized with the conjugate according to 
the method of Green et al. (17). The anti-G.sub.s .alpha. serum was 
desalted using Sephadex G-25 and the G class of immunoglobulins (IgG) was 
purified by HPLC using a Protein A affinity column (Rainin Instr. Co., 
Woburn, Mass.). 
Purified liver plasma membranes were solubilized on ice for one hour in 20 
mM Tris, 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 0.9% sodium cholate (pH 
8.0). The supernatant was collected after centrifugation at 13,000.times.g 
for five minutes at 4.degree. C. Solubilized plasma membranes were 
resolved by SDS polyacrylamide gel electrophoresis (12.5% acrylamide, 
0.051% N,N'-diallyltartatdiamide (DATD)) and transferred to Immobilon-P 
PVDF membranes (Millipore Corp., Bedford, Mass.) (19,21). Following the 
procedure of Mumby (20), the PVDF membranes were probed with G.sub.s 
.alpha. IgG and the bands were detected with .sup.125 I-labeled goat 
anti-rabbit IgG (1.0.times.10.sup.6 cpm/ml). The membranes were washed, 
blotted dry and exposed to Kodak XAR film with intensifying screens 
overnight. 
In performing the Western blot assays, protein was carefully measured in 
the liver plasma membrane supernatant to assure that equal amounts of 
soluble membrane protein would be loaded from the respective treatment 
groups. The membrane preparations were blotted for the presence of G.sub.s 
.alpha. to test for differences in expression level of the G protein that 
couples glucagon receptors to adenylyl cyclase in liver membranes. The 
Western blot data obtained showed that G.sub.s .alpha. expression was 
significantly higher in membranes from fatty Zucker rats compared to their 
lean litter mates. Assuming increased complex formation of G proteins with 
glucagon receptors, this result could explain the increased proportion of 
receptors in the high afinity binding state in liver membranes from fatty 
rats compared to lean rats. Furthermore, while G.sub.s .alpha. expression 
was not altered by exogenous PP in lean rats, treatment of fatty Zucker 
rats with PP decreased hepatic expression of G.sub.s .alpha. to levels 
similar to that seen in lean rats. These results indicate that increased 
expression of G.sub.s .alpha. may be the explanation for the enhanced 
efficacy of glucagon in fatty Zucker rats in activating adenylyl cyclase 
to produce hepatic glucose. These results indicate that exogenous PP 
appears to correct this hyper-responsiveness by altering G protein 
expression in the liver. 
Screening of compounds for the ability to treat NIDDM by inhibiting the 
ability of glucagon to activate cAMP. Compounds can be screened for the 
ability to treat NIDDM by administering the compound to lean and fatty 
Zucker rats or to ob/ob mice, db/ob mice or other animals suitable as 
models of hyperglycemia, obesity and impaired insulin function and their 
lean counterparts and assaying the efficacy of the compound in inhibiting 
the expression of the alpha subunit of the G.sub.s protein in the liver 
cell plasma membrane, thereby inhibiting the ability of glucagon to 
activate cAMP, according to the experimental protocols set forth in the 
Examples herein. The plasma insulin and glucose levels of these animals 
can also be determined following administration of the compound to be 
screened. A compound shown by the methods taught herein to reduce the 
hyperglycemia and hyperinsulinemia in the animal models employed by 
inhibiting the expression of the alpha subunit of the G.sub.s protein in 
the liver cell plasma membrane and inhibiting the ability of glucagon to 
activate cAMP, thereby inhibiting hepatic glucose production, is 
determined to be a compound effective in treating NIDDM. 
Screening of compounds for the ability to treat NIDDM by inhibiting beta 
cell and pancreatic islet hypertrophy. Compounds can be screened for the 
ability to treat NIDDM by administering the compound to lean and fatty 
Zucker rats or to ob/ob mice, db/ob mice or other animals suitable as 
models of hyperglycemia, obesity and impaired insulin function and their 
lean counterparts and assaying the efficacy of the compound in binding the 
vagal nuclear complex and inhibiting the secretion of digestive enzymes by 
the exocrine pancreas, according to the experimental protocols set forth 
in the Examples herein. The plasma insulin and glucose levels of these 
animals can also be determined following administration of the compound to 
be screened. A compound shown by the methods taught herein to reduce the 
hyperglycemia and hyperinsulinemia in the animal models employed by 
binding the vagal nuclear complex and inhibiting secretion of digestive 
enzymes by the exocrine pancreas, thereby inhibiting beta cell and 
pancreatic islet hypertrophy, is determined to be a compound effective in 
treating NIDDM. 
Screening of compounds for the ability to treat NIDDM by enhancing insulin 
sensitivity and reversing the effects of neuropeptide Y. Compounds can be 
screened for the ability to treat NIDDM by administering the compound to 
lean and fatty Zucker rats or to ob/ob mice, db/ob mice or other animals 
suitable as models of hyperglycemia, obesity and impaired insulin function 
and their lean counterparts and assaying the efficacy of the compound in 
binding the arcuate nucleus in the hypothalamus, thereby potentiating the 
effect of leptin in reducing neuropeptide Y synthesis, according to the 
experimental protocols set forth in the Examples herein. The plasma 
insulin and glucose levels of these animals can also be determined 
following administration of the compound to be screened. A compound shown 
by the methods taught herein to reduce the hyperglycemia and 
hyperinsulinemia in the animal models employed by binding the arcuate 
nucleus in the hypothalamus and potentiating the effect of leptin in 
reducing neuropeptide Y synthesis, thereby enhancing insulin sensitivity 
and reducing the effects of neuropeptide Y, is determined to be a compound 
effective in treating NIDDM. 
Protocols for administration of PP or carboxyl terminal fragments of PP to 
humans diagnosed with NIDDM. To treat NIDDM in a human subject, a protocol 
for human administration modeled after the one used by Bernston et al. 
(22) can be used. For example, between 2 and 500 .mu.g/kg body weight/day 
of PP and preferably between 4 and 100 .mu.g/kg body weight/day of PP can 
be intravenously infused into a subject over two 90 minute periods per day 
for about two days or until the symptoms of NIDDM, e.g., hyperglycemia and 
hyperinsulinemia, subside, either alone or in combination with insulin or 
other oral hypoglycemic agents administered in dosages known to be 
effective in reducing blood glucose levels. 
Alternatively, between 20 and 5,000 .mu.g/kg body weight/day of the 
carboxyl terminal fragment of PP and preferably between 40 and 1,000 
.mu.g/kg body weight/day of the carboxyl terminal fragment of PP can be 
intravenously infused into a subject over two 90 minute periods per day 
for about two days or until the symptoms of NIDDM, e.g., hyperglycemia and 
hyperinsulinemia, subside, either alone or in combination with insulin or 
other oral hypoglycemic agents administered in dosages known to be 
effective in reducing blood glucose levels. 
If analogs of PP or of the carboxyl terminal fragment of PP containing 
D-amino acid substitutions are available, between 0.5 and 50 .mu.g/kg body 
weight/day of PP analog or between 1 and 1,000 .mu.g/kg body weight/day of 
PP carboxyl terminal fragment analog can be intravenously infused into a 
subject over two 90 minute periods per day for about two days or until the 
symptoms of NIDDM, e.g., hyperglycemia and hyperinsulinemia, subside, 
either alone or in combination with insulin or other oral hypoglycemic 
agents administered in dosages known to be effective in reducing blood 
glucose levels. 
When PP or carboxyl terminal fragments of PP are administered in 
combination with insulin or other hyperglycemic compounds, the dosage 
range for PP can be from 1 to 100 .mu.g/kg body weight/day and from 10 to 
1,000 .mu.g/kg body weight/day for carboxyl terminal fragments of PP. 
The subject's plasma glucose and insulin levels can be measured according 
to protocols standard in the art. For example, a glucose tolerance test 
can be performed for each subject before the first infusion, during the 
last infusion and 24 hours after the last infusion. Levels of insulin, C 
Peptide, PP, glucagon and blood glucose as well as body weight can also be 
monitored throughout the period before, during and after infusion, 
according to protocols standard in the art. 
In addition, PP, the carboxyl terminal fragment of PP or D-amino acid 
analogs of these compounds can be administered as a subcutaneous infusion 
given twice a day. Preliminary dose studies would be carried out by 
starting with a dosage of 2 .mu.g/kg and doubling this dosage until the 
optimal dosage for a given subject was determined as indicated by 
measuring the subject's glucose tolerance and insulin secretion levels. 
After the optimal daily dosage is determined, the subject can receive 
injections, for example, twice daily for two or three days. The optimal 
daily dosage, once determined, can be administered daily or intermittently 
as needed, according to the subject's blood glucose and insulin levels. 
Levels of insulin, C Peptide, PP, glucagon and blood glucose levels can be 
monitored throughout the period before, during and after infusion, 
according to protocols standard in the art. 
Throughout this application various publications are referenced. The 
disclosures of these publications in their entireties 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 
1. Bray, G. A. 1977. The Zucker-fatty rat: a review. Fed Proc. 36:148-153. 
2. Gettys, T. W., P. M. Burrows and D. M. Henricks. 1986. Variance 
weighting functions in radioimmunoassay calibration. Am. J Physiol 
251:E357-E361. 
3. Gettys, T. W., R. Garcia, K. Savage, D. C. Whitcomb, S. Kanayama and I. 
L. Taylor. 1991. Insulin sparing effects of pancreatic polypeptide in 
congenitally obese rodents. Pancreas 6:46-53. 
4. Gettys, T. W., K. Okonogi, W. C. Tarry, J. Johnston, C. Horton and I. L. 
Taylor. 1990. Examination of relative rates of cAMP synthesis and 
degradation in crude membranes of adipocytes treated with hormones. Second 
Messengers and Phosphoproteins 13:37-50. 
5. Bray, G. A. and D. A. York. 1979. Genetically transmitted obesity in 
rodents. Physiol. Rev. 51:598-646. 
6. Offermanns, S., R. Schafer, B. Hoffman, E. Bombien, K. Spicher, K. D. 
Hinsch, Schultz and W. Rosenthal. 1990. Agonist-sensitive binding of a 
photoreactive GTP analog to a G-protein alpha-subunit in membranes of 
HL-60 cells. FEBS Lett. 260:14-18. 
7. Offermans, S., G. Schultz and W. Rosenthal. 1991. Identification of 
receptor-activated G proteins with photoreactive GTP analog alpha-32P!GTP 
azidoanilide. Methods Enzymol 195:286-301. 
8. Kasiske, B. L., M. P. O'Donnell and W. F. Keane. 1992. The Zucker rat 
model of obesity, insulin resistance, hyperlipidemia and renal injury. 
Hypertension 19 Suppl. I:I110-I115. 
9. Bray, G. A. 1992. Pathophysiology of obesity. Am. J. Clin. Nutr. 55 
Suppl. 488S-494S. 
10. Gettys, T. W. and J. D. Corbin. 1989. The protein kinase family of 
enzymes. In: Receptor Phosphorylation. V. K. Moudgil, editor. CRC Press, 
Boca Raton, Fla. pp.39-88. 
11. Gettys, T. W., T. A. Fields and J. R. Raymond. 1994. Selective 
activation of inhibitory G protein alpha-subunits by partial agonists of 
the human 5-HT1A receptor. Biochemistry 33:4283-4290. 
12. Lin, M. D., D. E. Wright, V. J. Jruby and M. Rodbell. 1975. 
Structure-function relationships in glucagon: properties of highly 
purified Des-His.sup.1 -, monoiodo-, and Des-Asn.sup.28, Thr.sup.29 ! 
(homoserine lactone.sup.27)-glucagon. Biochemistry 14:1559-1563. 
13. Martin, E. W. (ed.) Remington's Pharmaceutical Sciences, latest edition 
Mack Publishing Co., Easton, Pa. 
14. Eldridge et al. 1989. Cur. Topics in Microbiol. and Immunol., 
146:59-65. 
15. Oka et al. 1990. Vaccine, 8:573-576. 
16. Lynch, C. J., P. F. Blackmore, E. H. Johnson, R. L. Wange, P. K. Krone 
and J. H. Exton. 1989. Guanine nucleotide binding regulatory proteins and 
adenylate cyclase in livers of streptozotocin- and BB/Wor-diabetic rats. 
J. Clin. Invest. 83:2050-2062. 
17. Green, N., H. Alexander, A. Olson, S. Alexander, T. M. Shinnick, J. G. 
Sutcliffe and R. A. Lerner. 1982. Immunogenic structure of the influenza 
virus hemagglutinin. Cell 28:477-487. 
18. Raymond, J. R., C. L. Olsen and T. W. Gettys. 1993. Cell-specific 
physical and functional coupling of human 5-HT1A receptors to inhibitory G 
protein alpha-subunits and lack of coupling to G.sub.s -alpha. 
Biochemistry 32:11064-11073. 
19. Gettys, T. W., V. Ramkumar, R. J. Uhing, L. Seger and I. L. Taylor. 
1991. Alterations in mRNA levels, expression and function of GTP-binding 
regulatory proteins in adipocytes from obese mice (C57BL/6J-ob/ob). J. 
Biol. Chem. 266:15949-15955. 
20. Mumby, S., I.-K. Pang, A. G. Gilman and P. C. Sternwiess. 1988. 
Chromatographic resolution and immunologic identification of the alpha-40 
and alpha-41 subunits of guanine nucleotide-binding regulatory proteins. 
J. Biol. Chem. 263:2020-2026. 
21. Uhing, R. J., P. G. Polakis and R. Snyderman. 1987. Isolation of 
CTP-binding proteins from myeloid HL-60 cells. J. Biol. Chem. 
262:15575-15579. 
22. Bernston, G. G., W. B. Zipf, T. M. O'Dorisio, J. A. Hoffman and R. E. 
Chance. 1993. Pancreatic polypeptide infusions reduce food intake in 
Prader-Willi syndrome. Peptides 14:497-503. 
23. Sanacora G, Kershaw M. Finkelstein J A, White J D. 1990. Increased 
hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in 
genetically obese Zucker rats and its regulation by food deprivation. 
Endocrinology 127: 730-737. 
24. White J D, Olchovsky D, Kershaw M, Berelowitz M, Berelowitz M. 1990. 
Increased hypothalamic content of preproneuropeptide-Y messenger 
ribonucleic acid in streptozotocin-diabetic rats. Endocrinology 
126:765-772. 
25. Brady L S, Smith M A, Gold P W, Herkenham M. 1990. Altered expression 
of hypothalamic neuropeptide Y mRNAs in food-restricted and food-deprived 
rats. Neuroendocrinology 52:441-447. 
26. Chau S C, Leibel R L, Hirsch J. 1991. Food deprivation and age modulate 
neuropeptide gene expression in the murine hypothalamus and adrenal gland. 
Mol. Brain Res. 9:95-101. 
27 O'Shea R D, Gundlach A L. 1991. Preproneuropeptide Y messenger 
Ribonucleic acid in the hypothalamic arcuate nucleus of the rat is 
increased in food deprivation or dehydration. J. Neuroendocrinol. 3:11-14. 
28. Dean R G, White B D. 1990. Neuropeptide Y expression in rat brain: 
Effects of adrenalectomy. Neurosci. Lett. 114:339-344. 
29. Beck B, Stricker-Krongrad A, Burlet A, Nicolas J-P, Burlet C. 1990. 
Influence of diet composition on food intake and hypothalamic neuropeptide 
Y (NPY) in the rat. Neuropeptides 17:197-203. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
AlaProLeuGluProValTyrProGlyAspAsnAlaThrProGluGln 
151015 
MetAlaGlnTyrAlaAlaAspLeuArgArgTyrIleAsnMetLeuThr 
202530 
ArgProArgTyr 
35 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
LeuThrArgProArgTyr 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
LeuThrArgProArgTyr 
15 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
LeuThrArgProArgTyr 
15 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: Not Relevant 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
LeuThrArgProArgTyr 
15 
__________________________________________________________________________