Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR TREATING INSULIN RESISTANCE
THROUGH HEPATIC NITRIC OXIDE
Field of the Invention
The present invention relates to a compound and method for the
treatment of insulin resistance.
Background of the Invention
Patients with non-insulin dependent diabetes mellitus (NIDDM)
show insulin resistance, impaired glucose tolerance, and parasympathetic
neuropathies. Several other disease states are also associated with the co-
existence of parasympathetic neuropathies and insulin resistance. These
conditions include patients with chronic essential hypertension, obesity,
patients with liver disease, and patients with transplanted livers.
Chap et al., (15) demonstrated that the absorption of orally
administered glucose in conscious dogs was suppressed and delayed by
administration of atropine. The mechanism of this response was
demonstrated using an isolated, jointly perfused small bowel and liver
preparation in rats (19). Administration of insulin into the portal blood
supply
led to a parasympathetic nerve-mediated increase in absorption of glucose
from the lumen of the intestine. The effect could be blocked by atropine and
mimicked by carbachol. The -afferent limb of the reflex is activated by
insulin
with receptors located in the portal vein or liver and the efferent limb
represents muscarinic nerves supplying the intestine.
The neural pathway connecting the sensory and effector
branches of the reflex is not known but, in this unique preparation, could
only
occur through two sources. One route is from the liver along the portal vein
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through the posterior hepatic plexus to the intestine. The other involves
transmission through the celiac ganglion which remained intact in this
preparation. Regardless of the course, this is an example of a splanchnic
reflex that does not pass through the central nervous system. This
S mechanism likely serves the function of assuring that maximum glucose
absorption only occurs at a time when the organs sensitive to insulin-induced
uptake have also been stimulated.
In 1993, it was (10) first noted that the hypoglycemic response
to a bolus administration (5 minute infusion) of insulin (100 mU/kg i.v.) was
reduced by 37% by hepatic denervation in fasted cats. These fasted cats
developed insulin resistance immediately following acute denervation of the
liver. The degree of reduction of response to insulin was maximal after
anterior plexus denervation and did not increase further with addition of
denervation of the posterior nerve plexus or bilateral vagotomy thus
demonstrating that all of the nerves of relevance were in the anterior plexus.
To avoid the complexity of the reaction to hypoglycemia, a new
rapid insulin sensitivity test (R1ST) was developed (11) wherein a euglycemic
clamp was used following the administration of insulin and the response was
quantitated as the amount of glucose required to be infused over the test
period in order to hold arterial blood glucose levels constant. The RIST
methodology has been published in detail (11) and has been demonstrated in
both cats and rats. It is highly reproducible with up to five consecutive
responses being obtainable in cats and four in rats with blood glucose, levels
returning to control levels between each test. Insulin, glucagon, and
catecholamine levels remain unchanged between tests.
Cats showed a dose-related development of insulin resistance
using atropine (27): that was of a similar magnitude to that produced by
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surgical denervation. The dose of atropine required to produce a full insulin
resistance is 3 mg/kg (4 pmol/kg) administered into the portal vein. A similar
degree of insulin resistance was achieved with 10-7 mmol/kg of the M,
muscarinic selective antagonist, parenzepine, and with 10-6mmolkg of the M2
selective antagonist, methoctramine. Although not conclusive, the data
suggest that the response may be mediated by the M, muscarinic receptor
subtype (21).
Although the liver was clearly the organ that produced the
insulin resistance, it was not clear that the liver was the resistant organ.
In
order to determine the site of insulin resistance, a further series was done
in
cats that measured arterial-venous glucose responses across the hindlimbs,
extrahepatic splanchnic organs, and liver (22). The intestine was
unresponsive to the bolus insulin administration both before and after
atropine
or anterior plexus denervation or the combination of both. The hepatic
response was also not notably altered whereas the glucose uptake across the
hindlimbs, primarily representing skeletal muscle uptake, was decreased
following atropine or hepatic parasympathetic denervation. These results
indicated that interference with hepatic parasympathetic nerves led to insulin
resistance in skeletal muscle.
It was further demonstrated that the same degree of resistance
could be produced by pharmacological blockade of parasympathetic nerve
function using the muscarinic receptor antagonist, atropine (27, 28, 29, 30).
Also, the ability to reverse
insulin resistance produced by interruption of hepatic parasympathetic nerve
function and to reverse insulin resistance in a model of liver disease led to
the
issue of U.S. Patent 5,561,165 on October 1, 1996. Accordingly, it was
determined that, following a meal, insulin is released from the pancreas. The
presence of insulin in the blood elicits a hepatic parasympathetic reflex that
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results in the release of acetylcholine in the liver that results in the
generation
and release of nitric oxide which, in turn, results in the release of an
active
hormone (hepatic insulin sensitizing substance (HISS)) into the blood. HISS
controls the sensitivity of skeletal muscle to insulin so that in its
presence, the
muscle (and probably other tissues) is extremely sensitive to the effects of
insulin and results in a rapid uptake and storage of glucose.
In the absence of HISS, the large muscle mass is highly
resistant to insulin and the glucose storage in skeletal muscle is severely
reduced. Interruption of any part of the parasympathetic-mediated release of
HISS results in insulin resistance. This parasympathetic reflex regulation of
HISS release is the fundamental mechanism by which the body regulates the
responsiveness to insulin and this mechanism is adjusted according to the
prandial state, that is, according to how recently there has been a
consumption of nutrients.
In a fasted condition, HISS release in response to insulin is
minimal or absent so that if insulin is released in this situation, there is a
minimal metabolic effect. Following a meal, the parasympathetic reflex
mechanism is amplified so that HISS release occurs and results in the
majority of the ingested glucose being stored in skeletal muscle.
The consequence of lack of HISS release is the absence of
HISS results in severe insulin resistance. In this situation, the pancreas is
required to secrete substantially larger amounts of insulin in order that the
glucose in the blood is disposed of to prevent hyperglycemia from occurring.
If this condition persists, insulin resistance will progress to a state of
type 2
diabetes (non-insulin dependent diabetes mellitus) and eventually will lead to
a complete exhaustion of the pancreas thus requiring the patient to resort to
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injections of insulin. Thus, any condition in which the hepatic
parasympathetic reflex is dysfunctional will result in insulin resistance.
There is evidence that the insulin resistance that is seen in a
variety of conditions (non-insulin dependent diabetes, essentially
hypertension, obesity, chronic liver disease, fetal alcohol effects) results
from
a hepatic parasympathetic dysfunction. Lack of HISS would also be
anticipated to result in obesity at the early stage of the resultant metabolic
disturbance (the obese often become diabetic).
Normally after a meal, the liver takes up a small proportion of
glucose and releases HISS to stimulate skeletal muscle to take up the
majority of the glucose load. In the absence of HISS, the skeletal muscle is
unable to take up the majority of glucose thus leaving the liver to
compensate.
The hepatic glycogen storage capacity is insufficient to handle all of the
glucose, with the excess being converted to lipids which are then
incorporated into lipoproteins and transported to adipose tissue for storage
as
fat. Provision of HISS to these individuals would restore the nutrition
partitioning so that the nutrients are stored primarily as glycogen in the
skeletal muscle rather than as fat in the adipose tissue.
A major finding of direct relevance for designing therapeutic
approaches was that acetylcholine infused directly into the portal vein (2.5
gg/kg/min) resulted in a complete reversal of the insulin resistance induced
by
surgical denervation. Administration of the same dose of acetylcholine
intravenously produced no reversal. Intraportal administration directly
targets
the liver whereas intravenous infusion bypasses the liver and is not organ
selective. This demonstration is extremely important in that the data indicate
that the signal from the liver to skeletal muscle is blood-borne. This blood-
borne signal is referred to as the hepatic insulin sensitizing substance
(HISS).
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However, there has been no evidence of compounds which can be used to
control or alter this pathway.
It would, therefore, be useful to determine methods and
compounds for reversing insulin resistance by affecting the insulin resistance
pathway.
Summary of the Invention
According to the present invention, there is provided a method
of increasing insulin sensitivity by administering an effective amount of a
compound which stimulates nitric oxide production in the liver. Also provided
is a pharmaceutical composition having an effective amount of a compound
which stimulates nitric oxide production in the liver and a pharmaceutically
acceptable carrier.
Brief Description of the Drawings
Other advantages of the present invention will be readily
appreciated as the same becomes better understood by reference to the
following detailed description when considered in connection with the
accompanying drawings wherein:
Figure 1 is a bar graph showing the rapid insulin sensitivity test
(RIST) index before and after intravenous L-NAME administration and two
hours after administration;
Figure 2A and 2B are graphs showing (A) the control RIST
index versus the change from control after L-NAME administration; and (B)
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the control RIST index versus the change from control after
parasympathectomy and intraported atropine administration;
Figure 3 is a bar graph showing the RIST index in a control,
after intraportal or intravenous L-NAME administration, and after intraportal
atropine administration;
Figure 4 is a bar graph showing the RIST index in control, after
parasympathetic denervation, and after intraportal L-NMMA administration;
Figure 5 is a bar graph showing the RIST index in a control, and
after intravenous L-NAME and intraportal L-arginine administration;
Figure 6 is a bar graph showing the RIST index in a control,
after intraportal L-NMMA administration and two hours post L-NMMA
administration;
Figure 7 is a bar graph showing the RIST index in a control and
after intraportal L-NMMA and intraportal SIN-1 administration;
Figure 8 is a bar graph showing the RIST index in a control and
after intraportal L-NMMA and intraportal SIN-1 administration; and
Figure 9 is a bar graph showing the RIST index in a control,
after parasympathetic denervation, and after intraportal SIN-1 administration.
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Detailed Description
Generally, the present invention provides a compound and
method of increasing insulin sensitivity by administering an effective amount
of a compound which stimulates nitric oxide production in the liver. More
specifically, the compound can be administered as a nitric oxide donor or as a
stimulus that generates nitric oxide within the liver. Therefore, this
compound
and method can be useful in treating obesity, insulin resistance, and other
diseases associated with insulin resistance.
The compounds of the present invention can be considered,
generally, as members of the groups of nitric oxide agonists and NO donors.
Examples of such compounds include, but are not limited to: 3-
morpholinosyndnonimine (SIN-1), sodium nitrite, nitroprusside, S-nitroso-N-
acetyl-D, L-penicillamine (SNAP).
It was recently demonstrated that there is a powerful hepatic
parasympathetic reflex in response to insulin. Insulin results in a hepatic
parasympathetic activation of cholinergic muscarinic receptors which lead to
release of a hepatic insulin sensitizing substance (HISS) that enters the
bloodstream and regulates insulin sensitivity in skeletal muscle. Virtually
all of
the variability in insulin sensitivity in fed rats is demonstrated to be due
to
variability in the hepatic parasympathetic-dependent insulin response. Insulin
resistance is produced by surgical or pharmacological blockade of the hepatic
parasympathetic nerves and is easily demonstrated using a new insulin
sensitivity test. The insulin resistance so produced does not affect the
splanchnic organs but appears to be restricted to skeletal muscle and,
therefore, strongly resembles the sort of insulin resistance seen in non-
insulin-dependent diabetes mellitus and in patients with chronic liver
disease.
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Insulin resistance produced by surgical denervation of the liver
or the chronic bile duct ligation model of liver disease can be restored
completely to normal levels by intraportal but not intravenous administration
of acetylcholine. It is shown that many forms of insulin resistance in
different
disease states are secondary to hepatic parasympathetic neuropathy. This
pathway shows an unexpected but major role for hepatic parasympathetic
nerves in physiological and pathological regulation of glucose metabolism.
A recent series of studies reported that insulin initiates a
parasympathetic reflex which results in the release of acetylcholine (Ach) in
the liver (26-29). Ach acts on muscarinic receptors and causes the release of
a hepatic insulin sensitizing substance (HISS). HISS enters the blood and
sensitizes the skeletal muscle response to insulin. Since many cholinergic
effects are mediated through nitric oxide (NO), the hypothesis that this
parasympathetic effect is also mediated through NO (Figure 1) was tested.
To quantify insulin sensitivity in rats a modified euglycemic
clamp method for conducting a rapid insulin sensitivity test (RIST) (29) was
used. Interruption of the hepatic reflex response to insulin by surgical
denervation of the liver or atropine results in instant and reversible (26-28)
insulin resistance in skeletal muscle (27). To evaluate the involvement of NO,
two nitric oxide synthase (NOS) antagonists were used, N-nitro-L-arginine
methyl ester (L-NAME) and N-monomethyl-L-arginine (L-NMMA). The insulin
resistance produced by intravenous verses intraportal NOS antagonism was
also compared to determine if the liver was the site of NO action. 3-
morpholinosyndnonimine (SIN-1), a NO donor, was administered
intravenously or intraportally to reverse the insulin resistance produced by L-
NMMA.
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The results are consistent with the hypothesis that inhibition of
NOS in the liver interrupts the parasympathetic reflex, resulting in insulin
resistance and that NO delivered to the liver can restore insulin sensitivity
to
normal levels when insulin resistance is produced by blockade of NO
production in the liver or surgical destruction of hepatic nerves.
The normal response to insulin is a parasympathetic reflex
release of acetylcholine leading to nitric oxide generation and production of
HISS. Provision of nitric oxide to the liver can result in reversal of
parasympathetic neuropathy-induced insulin resistance regardless of the
cause of parasympathetic neuropathy. This includes situations where no true
neuropathy exists but where the primary dysfunction is with the ability to
produce nitric oxide. Nitric oxide can be administered to the liver by
provision
of nitric oxide donors or nitric oxide agonists or compounds that generate
nitric oxide within the liver when administered orally, intravenously,
intramuscularly, subcutaneously, or by delivery through a pump system
directly into the portal vein. Ideally such a compound would be administered
prior to a meal in order to restore normal hepatic parasympathetic responses
to insulin and thereby restore insulin sensitivity. The present invention
therefore provides a pharmaceutical composition containing an effective
amount of a compound which stimulates nitric oxide production in the liver
and a pharmaceutically acceptable carrier.
The above discussion provides a factual basis for the use of
compounds and methods for stimulating nitric oxide production in the liver for
treating insulin resistance. The methods used with the utility of the present
application can be shown by the following non-limiting examples and
accompanying figures.
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GENERAL METHODS
Standard molecular biology techniques known in the art and not
specifically described were generally followed as in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, New York (1989), and in Ausubel et al., Current Protocols in Molecular
Biology, John Wiley and Sons, Baltimore, Maryland (1989) and in Perbal, A
Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988),
and in Watson et al., Recombinant DNA, Scientific American Books, New
York and in Birren et al (eds) Genome Analysis: A Laboratory Manual Series,
Vols. 1-4 Cold Spring Harbor Laboratory Press, New York (1998) and
methodology as set forth in United States patents 4,666,828; 4,683,202;
4,801,531; 5,192,659 and 5,272,057.
Polymerase chain reaction (PCR) was carried out generally as in PCR
Protocols: A Guide To Methods And Applications, Academic Press, San
Diego, CA (1990). In-situ (In-cell) PCR in combination with Flow Cytometry
can be used for detection of cells containing specific DNA and mRNA
sequences (Testoni et al, 1996, Blood 87:3822.)
Delivery of therapeutics compound):
The compound of the present invention is administered and
dosed in accordance with good medical practice, taking into account the
clinical condition of the individual patient, the site and method of
administration, scheduling of administration, patient age, sex, body weight
and other factors known to medical practitioners. The pharmaceutically
"effective amount" for purposes herein is thus determined by such
considerations as are known in the art. The amount must be effective to
achieve improvement including but not limited to improved survival rate or
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more rapid recovery, or improvement or elimination of symptoms and other
indicators as are selected as appropriate measures by those skilled in the
art.
In the method of the present invention, the compound of the
present invention can be administered in various ways. It should be noted
that it can be administered as the compound or as pharmaceutically
acceptable salt and can be administered alone or as an active ingredient in
combination with pharmaceutically acceptable carriers, diluents, adjuvants
and vehicles. The compounds can be administered orally, subcutaneously or
parenterally including intravenous, intraarterial, intramuscular,
intraperitoneally, and intranasal administration as well as intrathecal and
infusion techniques. Implants of the compounds are also useful. The patient
being treated is a warm-blooded animal and, in particular, mammals including
man. The pharmaceutically acceptable carriers, diluents, adjuvants and
vehicles as well as implant carriers generally refer to inert, non-toxic solid
or
liquid fillers, diluents or encapsulating material not reacting with the
active
ingredients of the invention.
It is noted that humans are treated generally longer than the
mice or other experimental animals exemplified herein which treatment has a
length proportional to the length of the disease process and drug
effectiveness. The doses may be single doses or multiple doses over a
period of several days, but single doses are preferred.
The doses may be single doses or multiple doses over a period
of several days. Additionally, dosing can be single doses or multiple doses
prior to each meal for the duration of the disease. The treatment generally
has a length proportional to the length of the disease process and drug
effectiveness and the patient species being treated.
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When administering the compound of the present invention
parenterally, it will generally be formulated in a unit dosage injectable form
(solution, suspension, emulsion). The pharmaceutical formulations suitable
for injection include sterile aqueous solutions or dispersions and sterile
powders for reconstitution into sterile injectable solutions or dispersions.
The
carrier can be a solvent or dispersing medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable oils.
Proper fluidity can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required particle size in
the case of dispersion and by the use of surfactants. Non-aqueous vehicles
such a cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower
oil,
or peanut oil and esters, such as isopropyl myristate, may also be used as
solvent systems for compound compositions. Additionally, various additives
which enhance the stability, sterility, and isotonicity of the compositions,
including antimicrobial preservatives, antioxidants, chelating agents, and
buffers, can be added. Prevention of the action of microorganisms can be
ensured by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid, and the like. In many cases, it
will be desirable to include isotonic agents, for example, sugars, sodium
chloride, and the like. Prolonged absorption of the injectable pharmaceutical
form can be brought about by the use of agents delaying absorption, for
example, aluminum monostearate and gelatin. According to the present
invention, however, any vehicle, diluent, or additive used would have to be
compatible with the compounds.
Sterile injectable solutions can be prepared by incorporating the
compounds utilized in practicing the present invention in the required amount
of the appropriate solvent with various of the other ingredients, as desired.
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A pharmacological formulation of the present invention can be
administered to the patient in an injectable formulation containing any
compatible carrier, such as various vehicle, adjuvants, additives, and
diluents;
or the compounds utilized in the present invention can be administered
parenterally to the patient in the form of slow-release subcutaneous implants
or targeted delivery systems such as monoclonal antibodies, vectored
delivery, iontophoretic, polymer matrices, liposomes, and microspheres.
Examples of delivery systems useful in the present invention include:
Sharma, US Patent No. 5,225,182; Gyory et al, US Patent No. 5,169,383; Haak et
al, US Patent No. 5,167,616; Sanders et al., US Patent No. 4,959,217; Ranney,
US
Patent No. 4,925,678; Harris, US Patent No. 4,487,603; Ferrara, US Patent No.
4,486,194; Mayfield, US Patent No. 4,447,223; DeCant Jr., US Patent No.
4,447,224; and Higuchi, US Patent No. 4,439,196.
A pharmacological formulation of the compound utilized in the
present invention can be administered orally to the patient. Conventional
methods such as administering the compounds in tablets; suspensions,
solutions, emulsions, capsules, powders, syrups and the like are usable.
Known techniques which deliver it orally or intravenously and retain the
biological activity are preferred.
In one embodiment, the compound of the present invention can
be administered initially by intravenous injection to bring blood levels to a
suitable level. The patient's levels are then maintained by an oral dosage
form, although other forms 'of administration, dependent upon the patient's
condition and as indicated above, can be used. The quantity to be
administered will vary for the patient being treated.
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EXAMPLE 1
Methods and materials
Male Sprague-Dawley rates were fed ad-lib with standard
laboratory rat chow. The rats were anesthetized with an intraperitoneal
injection of phenobarbital sodium (65 mg/kg). Anesthesia was maintained
throughout the experiment by continuous infusion of pentobarbital solution
(1.0 ml/100g of body weight/hr, 1.0 mg/ml) through a cannula in the venous
side of the arterial-venous loop (described below). The temperature was
maintained at 37.5 0.5 C by means of a temperature controlled surgical
table and a heat lamp over the table. The body temperature was monitored
with a rectal probe thermometer (H18857, Hanna Instruments). The rats were
heparinized with 100 IU/kg heparin.
Surgical preparation. The left jugular vein was cannulated for
glucose infusion. Spontaneous respiration was allowed through a tracheal
tube. The blood samples (25 I) were obtained through a right femoral arterial-
venous loop (30). The right femoral artery was cannulated with the arterial
side of the loop. The right femoral vein was cannulated with the venous side
of the arterial-venous loop. Arterial blood pressure was monitored via the
arterial-venous loop by clamping the silicon sleeve on the venous side of the
loop. One of the advantages of using this loop is that blood samples can be
taken directly from a moving stream of blood with no need to wash or flush
sampling catheters. The arterial blood continuously flows through the loop
into the venous side. Intravenous infusions, except glucose, were given
through the venous side of the loop. After laparotomy, the portal vein was
cannulated with a 24G (OPTIVAT", Johnson & Johnson Medical Inc.)
intravenous catheter for intraportal drug administration.
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The rats were allowed to stabilize from the surgical interventions
for at least 30 minutes before any procedures were carried out. Arterial blood
samples were taken every five minutes, and glucose concentrations were
immediately analyzed by the oxidase method with a glucose analyzer (model
27, Yellow Springs Instrumentals) until three successive stable glucose
concentrations were obtained. The mean of these three concentrations is
referred to as the basal glucose level.
Rapid Insulin Sensitivity Test (RIST). After the basal glucose
level was determined, insulin (50 mU/kg in 0.5 ml saline) was intravenously
infused over five minutes. Euglycemia was maintained by a variable glucose
infusion. The glucose solution was prepared in saline (100 mg/ml) and
infused by a variable infusion pump (Harvard Apparatus). To avoid
hypoglycemia, the glucose infusion (5mg/kg/min) was started one minute
after insulin infusion. On the basis of the arterial glucose concentrations
measured at two minute intervals, the infusion rate of the glucose pump was
adjusted whenever required to clamp the arterial glucose levels as close to
the basal value as possible. The amount of glucose infused over 30 minutes
following insulin administration represents the magnitude of insulin
sensitivity
and is referred to as the RIST index. This method has previously been
described (30) and a standard operating procedure is given (11).
Rapid Insulin Sensitivity Test time controls. The control RIST
was repeated three times in the same animal (n=5). The rats were allowed to
stabilize between each RIST.
Rapid Insulin Sensitivity Test in control and after L-NAME at
doses 2.5 mg/kg and 5.0 mg/kg intravenously. After the control RIST, L-
NAME, at dose 2.5 mg/kg (n=12) or 5.0 mg/kg (n=17), was infused
intravenously over five minutes. A stable basal arterial glucose concentration
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was determined and a RIST was performed as described above. After 30
minutes of restabilization, basal arterial glucose concentrations were
determined and a second post L-NAME RIST was repeated to measure the
duration of action of each dose.
Rapid Insulin Sensitivity Test in control, after L-NAME
intravenously or intraportally and after Atropine. The RIST index was
determined before and after L-NAME (1.0 mg/kg) was infused either
intravenously (n=5) or intraportally (n=5) over five minutes. Atropine (3.0
mg/kg) was infused intraportally over five minutes and the RIST was
repeated.
Rapid Insulin Sensitivity Test in control, after surgical
denervation and after L-NMMA (n=3). After the control RIST, the nerve
bundles around the common hepatic artery were cut and the animal was
allowed to stabilize and the RIST was repeated. L-NMMA (0.73 mg/kg) was
intravenously infused and the RIST was performed.
Rapid Insulin Sensitivity Test in control, after L-NAME and after
L-arginine(n=6). After a control RIST, L-NAME (5 mg/kg) was infused
intravenously over five minutes. After the second RIST, L-arginine (50 mg/kg)
was infused intraportally and the RIST was repeated.
Rapid Insulin Sensitivity Test in control and after L-arginine
(n=4). After a control RIST, L-arginine (50 mg/kg) was infused intraportally
and insulin sensitivity was measured by the RIST.
Rapid Insulin Sensitivity Test in control and after L-NMMA (n=3).
After the control RIST, L-NMMA (0.73 mg/kg) was infused intraportally over
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five minutes. After the RIST, the animal was allowed to restabilize for 30
minutes. Basal arterial glucose concentrations were determined and a second
post L-NMMA RIST was repeated to measure the duration of the action of the
dose.
Rapid Insulin Sensitivity Test in control, after L-NMMA and after
SIN-I intraportally or intravenously. After the control RIST, L-NMMA (0.73
mg/kg) was infused intraportally over five minutes. After the RIST, SIN-1 (5.0
mg/kg) was infused either intraportally (n=5) or intravenously (n=4) over one
minute. Insulin sensitivity was measured by the RIST.
Rapid Insulin Sensitivity Test in control, after L-NMMA and after
intraportal SIN-1 (n=5). After the control RIST, L-NMMA (0.73 mg/kg) was
intraportally infused over five minutes. After the RIST, SIN-1 (10.0 mg/kg)
was
infused intraportally over two minutes and the RIST was repeated.
Rapid Insulin Sensitivity Test in control, after surgical
denervation and after intraportal SIN-1 (n=6). After the control RIST, the
nerve bundles around the common hepatic artery were cut and the animal
was allowed to stabilize. After the RIST, SIN-1 (10.0 mg/kg) was infused
intraportally over two minutes and the RIST was repeated.
Drugs. L-NAME, L-NMMA, L-arginine and atropine were
purchased from Sigma Chemical (St. Louis, MO). SIN-1 was purchased from
Alexis Corporation (San Diego, CA). The human insulin was obtained from Eli
Lilly & Company (Indianapolis, IN). All the chemicals were dissolved in
saline.
Data analysis. Data were analyzed using repeated-measures
analysis of variance followed by Tukey-Kramer multiple comparison test in
each group or, when applicable, paired and unpaired Student's t tests. The
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analyzed data were expressed as means SE throughout. Some results
were analyzed using linear regression analysis. Differences were accepted as
statistically significant at p<0.05. Animals were treated according to the
guidelines of the Canadian Council on Animal Care.
RESULTS
The index used to express insulin sensitivity is the total amount
of glucose (mg/kg) infused over 30 minutes after insulin (50 mU/kg)
administration in order to maintain euglycemia at the baseline level and is
referred to as the RIST index.
RIST in time controls. Three consecutive control RISTs were
performed in the same animal. The RIST indexes were 207.0 17.1 mg/kg,
202.4 25.7 mg/kg and 200.5 35.0 mg/kg, respectively. There was no
significant difference in glucose infusion between each RIST during the
experiment. The mean coefficient of variance (standard deviation/mean RIST
index for each rat) between the tests was 8.8 1.5%. The basal glucose
levels before each RIST (106.1 8.0 mg/dl, 99.4 10.8 mg/dl, 106.1 11.3
mg/dl, respectively) were not significantly different. The blood pressure was
stable (110 6.9 mmHg, 111.7 9.0 mmHg, 107.5 9.8 mmHg, respectively)
throughout each test. Thus, all three RISTs were similar.
RIST after intravenous L-NAME infusion. The control RIST
index was 178.5 16.5 mg/kg. L-NAME at dose 2.5 mg/kg (n=12)
significantly reduced the RIST index to 78.1 9.8 mg/kg and caused a 56.2
6.3 % inhibition of the control response. However after two hours when the
RIST was repeated again, the amount of glucose required to maintain the
euglycemia was 168.4 38.7 mg/kg which was not significantly different from
the control RIST (Figure 1). The blood pressure increased after L-NAME
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infusion from 107.6 4.7 mmHg to 133.4 5.3 mmHg but after two hours it
decreased to 110.4 10.7 mmHg. The basal glucose was similar before each
RIST (111.8 4.2 mg/ml, 90.4 5.0 mg/ml, 110.3 3.0 mg/ml, respectively).
In another set of animals (n=17), L-NAME at dose 5.0 mg/kg
significantly reduced the control RIST index (226.9 15.3 mg/kg) to 93.7
8.7 mg/kg and caused a 55.3 5.3% inhibition of the control response. Two
hours after administration, the RIST index was 75.8 16.0 mg/kg with 66.5
7.5% inhibition of the control response (Figure 1). After L-NAME infusion, the
blood pressure increased from 107.6 4.3 mmHg to 123.5 6.0 mmHg and
stayed at the same level, 120 .0 7.5 mmHg, after two hours. The basal
glucose was similar before each RIST (117.9 3.3 mg/ml, 107.4 3.4 mg/ml,
115.6 5.3 mg/ml, respectively). Thus both 2.5 mg/kg and 5.0 mg/kg L-
NAME produce similar insulin resistance but the duration of action is less
than
two hours with the low dose but was maintained for at least two hours for the
high dose.
The change from control after L-NAME, 2.5 mg/kg (n=12) and
5.0 mg/kg (n=17), was plotted against the control RIST index (mg/kg) (Figure
2a). The regression line has an x-intercept of 79.5 and a slope of 0.94
0.11. This relationship is interpreted to quantitate the HISS-dependent and
HISS-independent component of insulin action. Rats showing the greatest
response to insulin show the greatest HISS-dependent component of insulin
action.
FIST after intravenous verses intraportal L-NAME. The control
RIST index (n=5), of 224.1 23.5 mg/kg was not significantly reduced (177.9
21.2 mg/kg) after intravenous infusion of L-NAME (1.0 mg/kg). However
administration of atropine, a non-selective muscarinic antagonist,
intraportally
markedly reduced the RIST index to 95.3 14.6 mg/kg and caused a 56.0
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8.7% inhibition of the control RIST (Figure 3). The blood pressure was
constant throughout the experiment (96.0 4.5 mmHg in control, 100.0
11.5 mmHg after L-NAME and 93.0 8.6 mmHg after atropine). In the second
set of animals (n=5), the control RIST index (238.8 16.4 mg/kg) was
significantly reduced by intraportal L-NAME (1.0 mg/kg) (105.8 10.8 mg/kg),
causing a 54.9 5.2% inhibition of the control response. However,
administration of intraportal atropine caused a further significant reduction
in
RIST index (78.5 14.2 mg/kg) (Figure 3). The blood pressure increased
from 99.0 1.1 mmHg to 114.0 4.5 mmHg after L-NAME but it decreased to
104 8.0 mmHg after atropine consistent with data from the 2.5 mg/kg dose
showing effects wearing off by the time of the second (atropine) test. Thus,
intraportal but not intravenous L-NAME at the dose of 1.0 mg/kg caused
significant insulin resistance.
RIST after denervation and L-NMMA (n=3). Surgical
denervation of the hepatic anterior plexus significantly reduced the RIST
index from 228.3 13.8 mg/kg to 86.0 7.4 mg/kg and produced 62.0 4.8%
inhibition (Figure 4). Infusion of intraportal L-NMMA (0.73 mg/kg) did not
cause a further significant reduction in RIST index (80.8 10.5 mg/kg).
The change from control RIST index after intraportal atropine
(n=6) or hepatic denervation (n=10) plotted against control RIST index
(mg/kg) (Figure 2b) shows a x-intercept of 88.0 and a slope of 1.0 0.1.
Insulin's action has a parasympathetic-dependent and a parasympathetic-
independent component and the higher the RIST index is, the more the
response is inhibited by atropine or hepatic parasympathetic denervation.
RIST after L-NAME and L-arginine (n=6). After L-NAME (5.0
mg/kg, intravenous) the RIST index was significantly reduced from 237.0
26.1 mg/kg to 99.0 12.2 mg/kg and a 55.4 8.8% inhibition of control RIST
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was produced. L-arginine (50 mg/kg, intraportal) administration did not
reverse the inhibition by L-NAME (53.8 7.1 %) (Figure 5).
RIST after L-arginine. Following the control RIST, administration
of intravenous L-arginine (50 mg/kg, n=5) significantly inhibited the control
response by 48.8 8.2% (Figure 5).
RIST after L-NMMA(n=3). Administration of intraportal L-NMMA
(0.73 mg/kg) significantly reduced the RIST index from 236.8 37.6 mg/kg to
123.1 8.9 mg/kg (45.6 12.1% inhibition of the control RIST) (Figure 6).
The blood pressure was constant throughout the experiment (96.7 4.1
mmHg in control, 93.3 14.3 mmHg after L-NMMA before the RIST and 90.0
9.4 mmHg before the final RIST). After two hours RIST was repeated again
and the amount of glucose required to maintain the euglycemia was 76.1
14.8 mg/kg (65.1 13.0% inhibition of the control RIST). Thus, intraportal L-
NMMA produces insulin resistance that is maintained for two hours.
RIST after L-NMMA and SIN-1 intravenously or intraportally.
Intraportal infusion of L-NMMA (0.73 mg/kg, n=4) significantly reduced the
RIST index from 218.4 6.6 mg/kg to 88.4 21.6 mg/kg (59.6 9.7%
inhibition of the control RIST). Intravenous administration of SIN-1 (5.0
mg/kg)
did not reverse inhibition caused by L-NMMA (59.0 7.2% inhibition) (Figure
7). In the second set of animals (n=5), the control RIST index was 236.9
20.0 mg/kg. Intraportal infusion of L-NMMA (0.73 mg/kg) caused significant
insulin resistance and reduced the RIST index to 129.7 14.3 mg/kg and
caused (54.5 2.0% inhibition)(Fig. 7). Intraportal SIN-1 (5.0 mg/kg)
partially
reversed the inhibition caused by L-NMMA (24.0 11.6%). Thus, NO
production in the liver can partially reverse insulin resistance caused by NOS
antagonism.
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RIST after L-NMMA and intraportal SIN-1. Intraportal infusion of
L-NMMA (0.73 mg/kg, n=5) significantly reduced the RIST index from 221.34
30.9 mg/kg to 99.3 20.9 mg/kg (55.5 7.0% inhibition of the control
RIST). Intraportal SIN-1 (10.0 mg/kg) completly reversed the inhibition caused
by L-NMMA (0.6 5.8%) (Figure 8). Thus, higher NO production in the liver
can completely reverse insulin resistance caused by NOS antagonism.
RIST after denervation and intraportal SIN-1 (n=6). Surgical
denervation of the hepatic anterior plexus significantly reduced the RIST
index from 208.3 15.0 mg/kg to 87.7 10.3 mg/kg (56.4 6.7% inhibition of
the control RIST). Intraportal SIN-1 (10.0 mg/kg) completly reversed the
inhibition caused by denervation (3.8 10.4/%) (Figure 9). Thus, NO
production in the liver can reverse insulin resistance caused by surgical
denervation of the liver.
DISCUSSION
Previous studies (27-29) are consistent with the statement that
animals respond to insulin by activation of a hepatic parasympathetic reflex
release of a hepatic insulin sensitizing substance (HISS) that sensitizes
skeletal muscle to the effects of insulin. Surgical or pharmacological
ablation
of the hepatic parasympathetic nerves leads to insulin resistance.
Intraportal,
but not intravenous, Ach is capable of reversing the insulin resistance caused
by denervation. The hepatic parasympathetic reflex control of insulin action
is
mediated through hepatic NO and hepatic NOS antagonism and hepatic
denervation produce insulin resistance that is reversible by providing NO to
the liver using a NO donor. The parasympathetic reflex release of HISS is
concluded to be NO-mediated.
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Technical considerations. The rapid insulin sensitivity test
(RIST) is a modified euglycemic clamp method (11,30). Insulin (50 mU) is
infused over five minutes and the total amount of glucose infused (RIST
index) in order to maintain arterial glucose at the baseline level during the
30
minutes of the test is used to express insulin sensitivity in each test. The
difference between a control RIST and the RIST index after surgical hepatic
denervation or atropine is used to determine the hepatic parasympathetic
component of insulin action (27, 29). Three RISTs were performed, as time
controls, in the same rat during one experiment with a coefficient of variance
of 8.8 1.5%. The basal glucose levels before each RIST were not
significantly different. The blood pressure was stable throughout and between
each test. The RIST is sensitive and shows inhibition by L-NAME, L-NMMA,
atropine and hepatic denervation in anesthetized animals.
It had been shown that L-NAME is both a NOS inhibitor and a
muscarinic receptor antagonist (2). Although the mechanism or location of
action was not described, it was previously determined that L-NAME
produces insulin resistance that does not act through muscarinic antagonism
(22), thus indicating that both L-NAME and L-NMMA are suitable tools for the
present purpose.
Administration of intraportal L-NAME at 1.0 mg/kg causes
significant insulin resistance (22). In the present study those data are
confirmed using two additional doses of L-NAME, 2.5 mg/kg and 5.0 mg/kg.
Administration of L-NAME intravenously at 2.5 mg/kg and 5.0 mg/kg caused
significant and similar degrees of insulin resistance. However the effect of
the
low dose wore off within one hour whereas the high dose effect lasted for
more than two hours (Figure 1). An equimolar dose of L-NMMA to the dose
1.0 mg/kg of L-NAME had a duration of action of at least two hours (Figure 6).
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Nitric oxide synthase inhibition. Reports from other investigators
(1) suggest that inhibition of NOS by L-NMMA causes a reduction in skeletal
muscle perfusion and this has been suggested as the mechanism of insulin
resistance. In these experiments, intraportal L-NMMA (0.73 mg/kg) did not
result in hypertension (arterial pressure of 90 3.8 mmHg in control and 84.3
4.6 mmHg after L-NMMA), however significant insulin resistance occurred
(Figure 6). Oral administration of L-NAME caused hypertension but not insulin
resistance (26), showing that insulin resistance is not a result of vascular
effects but of a fundamental metabolic disorder. Surgical hepatic denervation
significantly reduced insulin sensitivity and subsequent NOS inhibition with L-
NMMA did not cause additional insulin resistance (Figure 4). If the NOS
antagonist effect was secondary to peripheral effects it should have been
additive to the effects of liver denervation. This observation shows that
hepatic parasympathetic interruption by surgery or NOS inhibition in the liver
caused insulin resistance by interruption of the same pathway.
To confirm the site of action of L-NAME intraportal infusion of L-
NAME dose (1.0 mg/kg) was compared with intravenous infusion of the same
dose. The intraportal, but not intravenous, dose caused significant insulin
resistance. The observation that L-NAME caused more insulin resistance
when administered intraportally (Figure 3) shows that the site of action of L-
NAME is the liver.
Insulin resistance caused by NOS antagonism is not a result of
reduction in skeletal muscle perfusion but rather is caused by blockade of the
parasympathetic reflex release of a hepatic factor that is released in
response
to insulin. This putative hepatic insulin sensitizing substance (HISS)
amplifies
the skeletal muscle response to insulin (28) and hepatic NOS inhibition
interrupts this pathway.
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Vasodilatory effect of insulin. Insulin-mediated vasodilation
increases glucose uptake in skeletal muscles (5,18,24). However, Scherrer
et at. (23) showed that L-NMMA, when infused into one arm, reduces forearm
blood flow and increases blood pressure, but does not alter the whole-body
glucose uptake (24). Natali et al. demonstrated that increasing forearm blood
flow with sodium nitroprusside in obese. hypertensive patients does not
improve insulin sensitivity (16). Hernandez Mijares et al. concluded that
after a mixed
meal, skeletal muscle blood flow does not increase enough for blood flow to
be a major contributor to glucose uptake (13). The effect of insulin on blood
flow is controversial. Some investigators report increased blood flow only at
high, supraphysiological insulin concentrations (19). Most investigators (1)
use the hyperinsulinemic euglycemic clamp technique to measure insulin
sensitivity. In this technique, insulin is infused at a constant rate for 2-3
hrs
before steady state conditions are achieved. It is possible that infusion of
insulin for long periods of time and at high concentrations results in
vasodilation and increased blood flow. However the insulin used in these
experiments, given over five minutes, is short acting and the RIST is
completed by 30 minutes. Baron et al. (1) report that during the
hyperinsulinemic euglycemic technique there is a fall in mean arterial
pressure caused by the vasodilatory effect of insulin. In these experiments
there was no significant change in blood pressure during insulin
administration. Furthermore, if NOS antagonism produced insulin resistance
secondary to direct blockade of dilatory responses to insulin in skeletal
muscle, the intravenous dose should have produced a greater effect than the
intraportal dose, the opposite of these findings (Figure 3). Similarly the
ability
of intraportal but not intravenous NO donor to reverse L-NMMA-induced
insulin resistance indicates that the drugs are acting through the liver.
Further,
if NOS antagonism produced insulin resistance secondary to blocking
vascular responses to insulin in skeletal muscle, the insulin resistance
caused
by hepatic denervation should have been made worse by the addition of this
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peripheral effect. Insulin resistance produced by denervation was not affected
by addition of a NOS antagonist. Thus, in these testing conditions the data
are consistent with insulin resistance following NOS antagonism being
secondary to a hepatic, rather than peripheral, effect.
Reversal of insulin resistance. L-arginine did not produce the
anticipated reversal of insulin resistance produced by L-NAME, but rather L-
arginine, by itself, caused insulin resistance (48.8 8.2%) (Figure 5). L-
NAME
not only blocks NOS but also blocks arginine uptake across the hepatocyte
plasma membrane (8). L-arginine is metabolized by NOS to NO, and by
arginase to urea and L-ornithine (6). Since the liver has a very high arginase
activity, most L-arginine administered is converted to L-ornithine by the
liver,
although L-arginine can reverse the vascular effects of L-NAME in the liver
(12). L-arginine also causes release of growth hormone (7,14) and glucagon;
both hormones reduce insulin sensitivity. This explains why insulin resistance
caused by L-NAME could not be reversed with L-arginine and why L-arginine
caused insulin resistance.
Reduction in blood flow to the nerves in diabetes leads to
neuropathy (3,4,9,17, 25) and has been shown to result from a decrease in
NO production in the vasculature (3, 9). Administration of L-NAME in normal
rats decreased nerve blood flow that was reversed by L-arginine (9,17). L-
NAME also caused basal vasoconstriction in the intestine that was reversible
by L-arginine (12).These observations show that L-arginine is capable of
reversing the effect of L-NAME in the vasculature. This shows that acute
insulin resistance caused by L-NAME is not secondary to effects on perfusion
of hepatic nerves or peripheral blood vessels since it was not reversed with L-
arginine.
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As an alternative to using L-arginine to reverse the effect of
NOS blockade, the NO donor, SIN-1, was used. Administration of intraportal,
but not intravenous, SIN-1 (5.0 mg/kg) partially reversed the insulin
resistance
caused by L-NMMA (Figure 7). However, administration of a higher dose of
SIN-1 (10.0 mg/kg) to the liver completely reversed the insulin resistance
caused by L-NMMA (Figure 8). This indicates that insulin resistance produced
after inhibition of NOS in the liver can be reversed by providing NO in the
liver. Also, administration of intraportal SIN-1 after denervation of the
liver
completely restored insulin sensitivity (Figure 9). Thus, NO production in the
liver is confirmed to be essential for insulin sensitivity.
Reversal of denervation-induced insulin resistance is additional
evidence that the parasympathetic reflex involves a hormonal pathway. If
there was a neural connection between the liver and skeletal muscle that was
controlling insulin sensitivity, this connection has been severed in order to
produce the insulin resistance. Administration of SIN-1 into the portal vein
cannot restore the response by a reflex pathway since the relevant nerves
have been cut.
HISS-dependent and -independent effect. The RIST index in
control responses and the reduction in control RIST index after atropine or
denervation was examined by linear regression as previously reported (29).
The rats showing the highest control RIST index had the greatest reduction in
response after atropine or denervation, and rats showing the lowest control
RIST index had the smallest decrease in control RIST index (Figure 4b). The
decrease in the RIST after denervation or atropine represents the HISS-
dependent component of insulin action. This shows a parasympathetic-
dependent component (to the right of the x-intercept) and a parasympathetic-
independent component (the x-intercept) of insulin action. A similar
relationship is observed after L-NAME administration. After L-NAME, the rats
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showing high control RIST indexes had large decreases in the RIST index,
and the rats showing small control RIST indexes had small decreases in the
RIST index (Fig 2a). This shows a hepatic NO-dependent component and a
NO-independent component involved in insulin action. The regression
analysis is not significantly different in slope or intercept using the
combined
atropine and denervation data compared to the NOS blockade data. There
is a parasympathetic-dependent and -independent and also a NO-dependent
and -independent component involved in insulin responsiveness and both
components act through the same pathway. This pathway is shown to consist
of an insulin-induced hepatic parasympathetic reflex, acting through
muscarinic receptors, resulting in production of NO in the liver, leading to
release of the putative hormone, HISS, that sensitizes the skeletal muscle to
the action of insulin. Interruption of this NO-mediated reflex inhibits HISS
release from the liver and insulin resistance follows.
In conclusion, there is a strong relationship between inhibition of
NOS in the liver and insulin resistance. Providing NO to the liver reverses
this
insulin resistance. Therefore, inhibition of the NOS in the liver interrupts
the
HISS pathway and, because HISS is needed to sensitize the skeletal muscle
response to insulin, insulin resistance occurs.
EXAMPLE 2
An insulin sensitivity test showing amount of glucose needed to
be administered after insulin (50 mU/kg i.v.) in order to maintain arterial
glucose steady is analyzed. In group 1, a nitric oxide synthase blocker
(blocks production of nitric oxide), L-NAME, was given into the portal vein
and
produced a 54.9 5.2% inhibition of insulin response. Atropine, in a dose
known to produce full blockade of the hepatic parasympathetic nerves, was
administered intravenously after L-NAME and produced a modest further
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resistance (67.2 4.9%). In a separate group of rats, the same dose of L-
NAME was given intravenously and did not produce significant insulin
resistance (19.8 7.5%). The blockade of muscarinic receptors with atropine
produced normal insulin resistance (56.0 8.9%) expected from
parasympathetic interruption. The data show conclusively that insulin
resistance produced by blockade of NO synthase did so by acting on the liver
rather than other tissues.
EXAMPLE 3
Insulin resistance (45.0 3.0% of normal response) is produced
by the blockade of nitric oxide synthase (eliminates production of nitric
oxide)
which is not reversed by administration of a nitric oxide donor intravenously
but is fully reversed by administration of the same dose directly to the liver
via
the portal vein. This response conclusively shows that the liver is the site
of
nitric oxide regulation of insulin sensitivity.
EXAMPLE 4
The hypothesis explaining hepatic parasympathetic reflex
release of hepatic insulin sensitizing substance (HISS) from the liver in
response to insulin, is that HISS is mediated by hepatic cholinergic receptors
and nitric oxide (NO) release. In absence of either nerve function or NO
releasem, severe insulin resistance occurs. Depending upon the pathology,
the resistance can be restored to normal by administration of a cholinergic
agonist or a source of nitric oxide.
Throughout this application, various publications, including
United States patents,_.are referenced by citation or number. Full citations
for
the publications are listed below.
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The invention has been described in an illustrative manner, and
it is to be understood that the terminology which has been used is intended to
be in the nature of words of description rather than of limitation.
Obviously, many modifications and variations of the present
invention are possible in light of the above teachings. It is, therefore, to
be
understood that within the scope of the appended claims, the invention may
be practiced otherwise than as specifically described.
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