The Effect of Vasopressin on Glucose Regulation
Condition: Insulin Resistance
MeSH terms: Insulin Resistance
Study type: Interventional

Summary: 
      Data from experimental animals and human epidemiological studies have suggested that
      hypohydration and/or low water intake is linked to poor glucose regulation and diabetes. The
      aim of this study is to investigate the effects of cellular dehydration on glucose in healthy
      non-diabetic individuals. METHODS: 60 males and females (30-55 y) will will undergo two
      experimental trials (ISO and HYP), consisting of a 2-h intravenous infusion of isotonic or
      hypertonic saline on two separate occasions, followed by a 4-h oral glucose tolerance test.
      Blood samples were taken from an antecubital vein in 30-min intervals starting at baseline
      for assessment of fluid and glucose regulating factors. Thirst will be assessed via visual
      analog following each blood sample. Energy substrate oxidation will be calculated via
      indirect calorimetry every 60 min.
    

Detailed description: 
      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.
    

Intervention name(s): Hypertonic Saline (3% NaCl); Isotonic Saline (0.9% NaCl)
Intervention type(s): Other; Other
Intervention description(s): 0.1 ml of 3% NaCl per kg of body weight per minute; 0.1 ml of 0.9% NaCl per kg of body weight per minute
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Single (Participant)
Primary purpose: Basic Science
Keywords: copeptin; vasopressin; dehydration; insulin resistance; Diabetes; Glucose regulation