Studies on the Adaptive Responses to Hypoxia
Condition: Hypoxia; Diabetes Mellitus, Type 1
MeSH terms: Diabetes Mellitus, Type 1; Hypoxia
Study type: Observational

Summary: 
      The general aim of this study is to define the response to hypoxic challenge in patients with
      diabetes. The investigation will provide response for different questions that are central
      for explaining the development of complications in diabetes

        -  have patients with diabetes an impaired reaction to adapt to hypoxia

        -  what consequence has hypoxia challenge on respiratory and on cardiovascular regulation
           in patients with diabetes

        -  what consequence has diabetes on the angiogenetic response to hypoxia
    

Detailed description: 
      Complications of diabetes represent the main concern for modern diabetes therapy, and it has
      become a priority to further characterise the pathophysiological mechanisms of these
      complications to ensure the development of novel rational therapeutic strategies.

      Although the prolonged exposure of tissues to hyperglycaemia is the primary causative factor
      for chronic diabetes complications, it has recently become increasingly evident that hypoxia
      also plays an important role in all diabetes complications . A low tissue concentration of
      oxygen in diabetes is the consequence of several mechanisms (e.g., deficient blood supply
      because of micro- and macro-vascular disease , poor local oxygen diffusion because of local
      oedema, or as the result of increased oxygen consumption).

      Adaptive responses of cells to hypoxia are mediated by Hypoxia-Inducible Factor 1 (HIF),
      which is a heterodimeric transcription factor that is composed of and subunits, which are
      both constitutively expressed in mammalian cells. The regulation of HIF activity is
      critically dependent on subunit degradation in normoxia. Under hypoxic conditions, HIF-1 is
      stabilised, binds to HRE (hypoxic responsive elements) and up-regulates a gene series that is
      involved in angiogenesis, glycolytic energy metabolism, cell proliferation, and survival,
      which enables cells to adapt to reduced oxygen availability. It is estimated that that more
      than 800 genes are direct HIF targets . HIF-1 is central for expression of multiple
      angiogenic growth factors (reviewed by[6]), endothelial progenitor cells (EPC) recruitment .
      Recently, it has been proposed that microRNAs (ex. mir210) also mediate some HIF-1 functions
      .

      Several pieces of evidence point to a defective response of diabetic tissues to hypoxia. An
      impaired hypoxia response is present in all tissues investigated in diabetic animal and
      diabetic patients. Hyperglycaemia directly represses HIF stability and function at multiple
      levels, a mechanism that is not completely understood.

      An impaired reaction to hypoxia in diabetes might have important consequences in acute
      hypoxic challenges as acute heart infarction, stroke, limb ischemia but also in subtle
      regulation of cardiovascular and respiratory system as a consequence of autonomic neuropathy
      with potential severe prognostic effect on late cardiovascular events . Interventional
      studies have addressed the cardiovascular responses to intermittent hypoxia (IH) compared
      with normoxia exposure in patients with diabetes . However in order to establish the
      appropriateness of the cardiovascular reaction to hypoxia in diabetes it is a need to
      investigate the cardiorespiratory responses towards IH in patients with diabetes compared
      with matched non diabetic control subjects. Moreover it is essentially to establish the
      angiogenetic response in the same experimental design. The hypothesis for this study is that
      both the cardiorespiratory reaction mediated through autonomic nervous system and
      angiogenetic response are impaired in patients with diabetes as a consequence of an impaired
      HIF reaction as seen in other tissues .

      The investigators plan to study the effect of intermittent hypoxia (IH) that will consists of
      five hypoxic periods (13% O2 inspired fraction of oxygen) each lasting 6 min, with five
      normoxic intervals of same duration in 15 patients with type 1 diabetes without clinical
      signs of neuropathy and in 15 non diabetic controls matched for sex, BMI, age. The study will
      be performed during one day.

      Methods:

      Baseline data will be obtained in the morning of each testing day at least 2 h after
      breakfast. Subjects are advised to abstain from caffeinated beverages for 12 h and from
      alcohol for 36 h prior to testing.

      Blood pressure and heart rate and arterial oxygen saturation will be continuously measured.
      In case of a decrease in oxygen saturation 80% or the occurrence of symptoms, hypoxia would
      have been discontinued until oxygen levels reached at least 80%. A technician will regulate
      and control the breathing periods under supervision of a medical doctor in a way that the
      intervention could not be observed by any patient. Thereafter, three measurement sessions
      will be performed: immediately after (t2), after 3 h (t3), and after 6 h (t4). After t2, each
      patient will obtaine an individual meal according to diet requirement.

      Cardiovascular and respiratory testing.

      Hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR) will be
      evaluated to determine respiratory system activity. All patients will be tested in the supine
      position in a silent room at comfortable temperature. Before participants will be connected
      to a rebreathing circuit through a mouthpiece with an antibacterial filter, spontaneous
      breathing of room air at rest will be performed for4 min in order to obtain baseline data.

      During each condition, the investigators will perform continuous measurement of oxygen
      saturation (SaO2) by a pulse oximeter and end-tidal CO2 (CO2-et) using a capnograph connected
      to a mouthpiece. Recordings of electrocardiogram will be performed by chest leads, and
      continuous noninvasive blood pressure will be recorded using the cuff method. A heated Fleish
      pneumotachograph will be connected to a differential pressure transducer and inserted in
      series to the expiratory component of the rebreathing system to measure airway flow. For
      measurement of the response to hypoxia, the participants will be connected to a rebreathing
      circuit inducing progressive decrease in SaO2 while maintaining CO2-et values at constant
      levels, until SaO2 will reach 80%, and measuring breath-to-breath changes in minute
      ventilation. The response to hypercapnia will be evaluated by ventilatory changes induced by
      progressive increase in CO2-et levels (up to 13 mmHg above resting levels), while SaO2 will
      be maintained 98% by oxygen at very low flow.

      Cardiovascular autonomic function will be determined performing four tests according to
      recent guidelines: deep-breathing, 30:15 ratio, Valsalva maneuver and systolic blood pressure
      response to standing. Cardiovascular autonomic neuropathy will be defined as the "presence of
      two or more abnormal tests".

      Measurement of chemoreflex sensitivity: The slope of the linear regression line of minute
      ventilation versus SaO2 or CO2-et indicates in each case the chemoreflex sensitivity to
      hypoxia or hypercapnia. In the hypercapnic test, the point at which the ventilation start to
      increase indicates as ventilator recruitment threshold to CO2 (VRT- CO2). VRT- CO2 will be
      identified by interpolating the ventilation/CO2-et plot by a fourth-order polynomial
      function.

      Assessment of baroreflex sensitivity: The baroreflex sensitivity (BRS) will be measured
      during spontaneous breathing at each measurement session. Since previous studies did not
      document a better performance of one method over the others, the investigators will calculate
      the average of seven different methods: positive and negative sequences, the a-coefficient in
      the low- and high-frequency bands and its average, the transfer function technique, and the
      ratio of SDs of R-R interval and systolic blood pressure variabilities. Besides BRS, SD of
      the R-R interval (SDNN) will be applied to determine a global index of heart rate
      variability. This selection is done based on the fact that normal distribution is more
      pronounced in this variable compared with other indices of variability (e.g., variance). The
      Hypoxic ventilatory response (HVR) and hypercapnic ventilatory response (HCVR), baroreflex
      sensitivity (BRS) will be evaluated before (t1), immediately after (t2), 3 h (t3), and 6 h
      (t4) after IH.

      The angiogenetic potential will be evaluated at the same endpoints by measuring in serum
      relevant cytokines that are gene targets for HIF-1 (i.e. VEGFA, SDF-1a, erythropoietin etc).
      The direct response of HIF signaling will be evaluated by the serum levels of mir210 which
      exclusively regulated by HIF.

      The absolute endothelial precoursor cell account (EPC) response will be evaluated at the same
      time points by FACS analysis of the number of CD34+/CD133+/KDR +
    

Intervention name(s): Hypoxia
Intervention type(s): Other
Intervention description(s): Intermittent hypoxia which consist of five hypoxic periods (13% O2 inspired fraction of oxygen) each lasting 6 min, with five normoxic intervals of same duration
Observational model: Case-Control
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Patients with diabetes are included from Department of endocrinology. Healthy controls are
        included via announcement och social media.
      
Biospecimen description: 
      whole blood, serum
    
Keywords: HIF; hypoxia; chemoreflex; baroreflex; MIR210