The Effect of Vasopressin on Glucose Regulation

Introduction The neurohypophysial hormone arginine vasopressin (AVP), also known as
antidiuretic hormone, was one of the first hormone identified for its vasopressin properties
in 1895 by Oliver and Schäfer. They showed that extract of pituitary gland increased blood
pressure in anesthetized dogs. AVP is mainly synthesized in the paraventricular and
supraoptic nucleus of the hypothalamus. The hormone is transferred to the neural lobe of the
posterior pituitary where it is released to the circulation. Target organs perceive the
hormonal stimuli by three different receptors: V1a, V1b and V2. The receptor V1a is mainly
expressed in the vascular wall and is responsible for vasoconstriction. The receptor V1b is
mainly found in the anterior pituitary, mediating the secretion of the adrenal corticotropin
hormone, while the V2 receptor is mainly expressed in nephron tubules triggering water
reabsorption. Since the discovery of AVP, both the vasopressin and antidiuretic properties
have been very well studied and documented.

Other than the AVP effects on blood pressure and water homeostasis, the hormone is
implicated in a variety of other functions including pain, bone and lipid metabolism,
hypertension, social behavior, aging, cognitive function, cellular proliferation,
inflammation, infections, homeostasis, hypothalamic-pituitary-adrenal axis, and diabetes.
All these effects could provide useful insight into many diseases. Therefore, the focus of
this application is on the effects of AVP on glucose regulation in healthy humans.

AVP is known to enhance hepatic glycogenolysis by activation of V1a receptors and by
increasing the release of glucagon, resulting in increased glucose levels in experimental
animals. Even when glucagon receptors in the liver are blocked, AVP still increases blood
glucose. The V1b receptors have been identified in both alpha and beta cells of the islets
of Langerhans. Thus, AVP stimulates insulin secretion counteracting the increase in blood
glucose. In an experiment with AVP V1a and V1b receptor knockout mice, alterations in
glucose and fat metabolism were observed, suggesting that AVP might play a role in glucose
regulation and metabolic disorders. Studies in humans with a genetic variation of AVP V1a
receptor showed increased prevalence of diabetes in overweight or subjects with high fat
diet. Recently, a study in rats prone to metabolic dysfunction, examined the effect of
long-term influence of vasopressin on glucose homeostasis. It reported that high vasopressin
enhanced hyperinsulemia and glucose intolerance in obese rats, while treatment with
vasopressin receptor V1a antagonist reduced glucose intolerance.

In a French epidemiological study, a cohort of 3,615 males and females with normal fasting
blood glucose was followed for 9 years. It indicated that water intake was inversely and
independently associated with the risk of developing hyperglycemia. The authors hypothesized
that their results were due to hypohydration related increase in plasma vasopressin. More
recently, a Swedish cohort of 2,064 subjects from the malmo diet and cancer study was
analyzed after 15.8 y with an oral glucose tolerance test. They found that copeptin (a
reliable and clinical surrogate marker of AVP) independently predicted diabetes mellitus and
abdominal adiposity.

Interestingly, hypohydration and low water drinking is linked to chronic elevated AVP. In a
recent study with free-living adults, low habitual water intake led to significantly
elevated AVP compared to adults with high water intake.

Experimental Design Sixty subjects between the age of 30 to 55 with absence of insulin
resistance will be recruited in the study. Thirty of the adults will be with normal body
mass index (BMI; 15 males and 15 females, 18.5 kg∙m-2 < BMI ≤25 kg∙m-2) and thirty
overweight or obese adults (15 males and 15 females, 28 kg∙m-2 ≤ BMI ≤35 kg∙m-2) will be
recruited to participate in the study. Sample size estimation showed that 60 subject will
provide power of 0.8 with alpha level set at 0.05. The effect of vasopressin on glucose
metabolism will be studied via osmotic stimulation of vasopressin (AVP) utilizing hypertonic
saline infusion followed by an oral glucose tolerance test. Each subject will perform two
identical experiments, differing only in the sodium chloride (NaCl) content of the infusion
(isotonic vs. hypertonic saline). All female subjects will perform both trials during their
early follicular phase, approximately 2-6 days post menses onset to ensure low endogenous
levels of estrogen and progesterone. After resting in a seated position for 30 min, subjects
will be infused intravenously either 3% NaCl (Hypertonic saline, hence HYPER) or 0.9 % NaCl
(Isotonic saline, hence ISO) over a 120 min period at an infusion rate 0.1 ml/min/kg of body
weight in a single-blind fashion and in counterbalanced order. This hypertonic saline
infusion increases plasma osmolality from 285 to at least 300 mmol/kg. From a separate
venous catheter, blood samples will be taken every 30 min. Following infusion, subjects will
rest for an equilibration period of 30 min before they start a 4-h oral glucose tolerance
test.

Subject preparation for oral glucose tolerance test (OGTT): Subjects should eat a normal
diet for 3 days prior to the test, and undertake normal physical activity. Dinner will be
standardized the day before and no alcohol will be allowed. Subjects will be instructed to
fast at least 10 h prior to the test.

Procedure for OGTT: The OGTT test will consist of a 75 g of glucose ingestion followed by
blood sampling period, with samples collected every 30 min for 240 min. Urine samples will
be collected at the end of the infusion and at the end of the OGTT. Blood pressure will be
recorded via an automated sphygmomanometer following each blood sample. Oxygen uptake and
respiratory exchange rate will be assessed hourly via indirect calorimetry, for a total of
seven times per trial. During blood sampling, thirst perception and mouth dryness will be
also assessed via visual analog scales.

Samples Analysis A total of fourteen blood samples will be collected in each experimental
trial and analyzed for: hematocrit (Hct), hemoglobin (Hb), osmolality (Osm), sodium (Na) and
potassium (K), total plasma protein, glucose, insulin, c-peptide, glucagon, copeptin, plasma
renin activity, corticotropin releasing hormone (CRF), cortisol, triglycerides and free
fatty acids (FFA).

Urine samples will be analyzed for osmolality and urine specific gravity fresh. Backup
samples of serum, plasma, and urine will be stored, frozen at -80°C, in the event additional
analysis are needed, or as replacements for broken vials.

Data Handling & Analysis To ensure quality and integrity of the data collected case report
forms will be utilized. The case report forms (CRF) will be designed to record data
collected in a way that will meet the highest standards. CRF will be developed, tested and
approved prior to any subject enrolment. The scientist involved in the data collection of
the study will be trained on the use of CRF prior to beginning of data collection. A paper
and digital library for all CRFs will be established and maintained during the experiment
and at least for 2 more years after the publication of the manuscripts. The data base
manager and the principal investigator will be the only person that will have access to
identifiable subject information. All the rest of the documents will be coded to ensure
subject anonymity. Data and Safety Monitoring Plan includes: Overall framework for data and
safety monitoring, responsible party for monitoring, and procedures for reporting adverse
events/unanticipated problems. Following data collection, data entry will take place by two
authorized scientists. Data from Quest diagnostics are available in PDF format and data
entry will be also performed and verified by two scientists. Data integration and database
cleaning will be performed in statistical software via analysis and visualization.

The primary response variable glucose metabolism will be captured by 4 variables (glucose,
insulin, C-Peptide and glucagon) all measured on a ratio scale, and on 14 occasions.

Secondary Outcomes will be: Hb, Hct, Total Proteins, Osmolality, Na, K, Copeptin, adrenal
corticosteropin releasing hormone (ACTH), CRH, Angiotensin II, plasma renin activity (PRA),
Aldosterone, Triglycerides, FFA.

Additional repeated continuous outcomes will be: (1) Blood pressure which will be measured
on 14 occasions; (2) Oxygen uptake and respiratory exchange rate which will be assessed on 7
separate occasions; and (3) thirst perception and mouth dryness which will be measured on 14
occasions.

Covariates Measurements Treatment groups (isotonic vs. hypertonic saline) will be the
primary comparison of interest. Other covariates will include sex (Females vs. males) and
weight status (normal weight vs. overweight/obese), age (30-55) and time (1-14).

To examine if the mean response profiles are similar in the groups, i.e. whether patterns of
change over time in the mean response vary by group, the present study will further explore
group by time interaction effects: (e.g. treatment by time interaction; weight by time
interaction; sex by time interaction).

Data Analysis Plan

- For this quasi-experimental with repeated measure-design, the researchers will conduct
a longitudinal analysis to describe changes in the mean response over time, and how
these changes are related to the covariates of interest.

- The normality of these continuous variables will be assessed by conducting the
Shapiro-Wilk test of normality.

- For all continuous outcomes, summary statistics (mean and standard deviations) will be
conducted at each time and by sequence. Moreover, correlations between glucose
metabolism measures will be performed.

- Percentages will be calculated for covariates that are measured on a nominal scale, and
mean and ± standard deviations presented for those measured on a continuous scale.

- The distributions of insulin sensitivity indexes and areas under the curve will be will
be assessed.

Statistical Modeling To study how changes in the mean response relate to covariates over
time, Generalized Linear Mixed Effects Modeling -with random intercepts and slopes- will be
employed using the Restricted Maximum Likelihood Estimation. It is assumed that each group's
mean response will change linearly over time. However, if the mean response over time is not
linear, higher-order polynomial trends will be explored.

The researcher will fit the appropriate covariance pattern model to account for the
correlations among the repeated measures so that appropriate inferences are made.

Statistical significance will be determined at an alpha of 0.05. All statistical analyses
would be carried out with the following statical software STATA©, JMP© or SAS©.

Anticipated Findings and Conclusions During the hypertonic saline infusion trial water
balance will be artificially manipulated (hypertonic hypervolemia). The increase in plasma
osmolality will stimulate vasopressin secretion. It is anticipated that AVP stimulation will
elevate insulin to a greater degree than glucose resulting in higher insulin resistance. It
is also expected that great urine osmolality as a response to elevated vasopressin levels
and lower urinary output.

Significance of the Project Diabetes along with obesity is one of the leading
non-communicable diseases in the developed and developing countries. More than 29 million
Americans are diabetic and another 86 million are in a pre-diabetic state. The cost of
diabetes in 2012 was $245 billion and it is growing. Hypohydration on the other side, is a
quite common phenomenon linked to many health issues like urinary tract infections, kidney
stones, cardiovascular diseases, mood state and cognitive performance. One of the potential
mechanisms behind the effects of hypohydration is the elevated level of AVP. Recent
epidemiological data and experiments in animals indicate that hypohydration and high
vasopressin are linked to both diabetes and glucose dysregulation. However, no experimental
data from a controlled trial in humans exist. The purpose of the proposed study is to
perform a controlled trial on healthy humans to examine the effect of elevated vasopressin
on glucose regulation.

Inclusion Criteria:

- Males or females of age 30-50 y old

- Signed Informed Consent prior to the initiation of any trial procedure

- Sedentary lifestyle

Exclusion Criteria:

- Body Mass Index (BMI) greater than 35 kg/m2, below 18.5 kg/m2, and between 25 and 28
kg/m2

- Surgical operation on digestive tract, except possible appendectomy

- Regular smoker within the past 6 months

- Diagnosed diabetes (Type I or Type II)

- Previous diagnosis of cardiovascular disease including hypertension

- Inability to participate in the entire study

- Drastic change in weight in the last month (more than 3 kg)

- Serotonin re-uptake inhibitors (i.e. Prozac)

- Impaired kidney or liver function

- Insulin therapy

- injectable contraceptives

- Currently taking medications that impair water balance

- Commuting by bike the day of the experiment

- Pregnancy