In Vivo Assessment of Cellular Metabolism in Humans
Condition: Normal Cellular Metabolism
Study type: Interventional
Phase: Phase 1

Summary: 
      This is a pilot study to establish an arterial venous methodology to measure the activity of
      the TCA cycle or flux directly in tissues of human beings. It will also perform correlative
      studies to study the proteome, metabolome, oxygen consumption, carbon dioxide production and
      exosomes derived from the arterial venous supply of tissues with correlation to the TCA cycle
      activity.
    

Detailed description: 
      The tricarboxylic (TCA) or Krebs cycle is the "central hub of cellular metabolism" that takes
      place within the mitochondria. It is a series of sequential chemical reactions that generate
      cellular energy in the form of ATP. In addition, the cycle provides intermediate metabolites
      that are utilized in the biosynthesis of amino acids and fatty acids as well as reducing
      agents such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide
      (FADH2) that are used in numerous biochemical reactions. The dysfunction of the TCA cycle is
      recognized for its association in neurodegenerative and cardiovascular diseases, metabolic
      syndromes, tumorigenesis and aging. Hence, being able to measure the activity or flux of the
      TCA cycle either in vitro or in vivo holds significant clinical significance. Almost all
      studies are based on in vitro approaches except NMRS based studies that involve multiple
      non-validated assumptions.

      Various stable isotope labeling studies have been used to estimate the TCA cycle flux by
      measuring one or more labelled intermediate metabolites within the cycle. Unfortunately,
      these labelled intermediates are often present through only partial segments of the cycle due
      to exchange, anaplerosis (entrance into the cycle), cataplerosis (export out of the cycle) or
      incomplete cycling. Though these previous isotope labeling studies of the TCA cycle flux were
      qualitatively informative, many were quantitatively inaccurate due to unexpected dilutions of
      the TCA cycle intermediates arising from unlabeled precursors.

      This is a pilot study to establish a novel methodology using mass-isotopomer flux analysis
      after infusions of 2-13C-Acetate, 2-15N-Glutamine and D5-phenylalanine to measure the in vivo
      TCA cycle flux in tissues of human beings. This study will simultaneously determine the
      validity of measuring the TCA cycle flux in tissue indirectly through dynamic differences in
      enrichment of labelled TCA cycle intermediates between arterial and venous blood supplies of
      that particular tissue bed (i.e. arteriovenous model or A-V balance technique). We propose to
      measure the rates of the different metabolic reactions within the TCA cycle by tracing the
      position-specific 13C and 15N transfer between the intermediate metabolites in order to
      characterize the oxidative, anaplerotic, cataplerotic and exchange rates across the TCA
      cycle. The use of 2-15N-Glutamine will specifically allow us to determine the rate of
      glutamine entry into the cycle via its conversion to glutamate, thus providing a more
      accurate quantification of the TCA flux.

      This methodology will be validated in the setting of controlled physiologic perturbations in
      human study participants such as low endogenous insulin levels alone or in combination with
      high glucagon levels.

      Finally, correlative studies evaluating the mitochondrial activity in the skeletal muscle
      tissue, the oxygen consumption in the skeletal and splanchnic tissue beds, the role of
      circulating exosomes derived from the arteriovenous circulation of the skeletal and
      splanchnic tissue beds and the changes in the whole body metabolome will also be performed:

        -  First, mitochondrial respiration will be measured by high resolution respirometry
           (Oxygraph, Oroboros Instruments, Innsbruck, Austria) using a stepwise protocol to
           evaluate various components of the electron transport system. Protein content of the
           mitochondrial suspension will be measured using a colorimetric assay (Pierce 660-nm
           Protein Assay). Oxygen flux rates will be expressed per tissue-wet weight and per
           milligram of mitochondrial protein.

        -  Secondly, reactive oxygen species (ROS) emissions will also be evaluated on all skeletal
           muscle tissue samples. Briefly, a Fluorolog 3 (Horiba Jobin Yvon) spectrofluorometer
           with temperature control and continuous stirring will be used to monitor Amplex Red
           (Invitrogen) oxidation in freshly isolated mitochondrial suspensions obtained from the
           skeletal muscle biopsies. Amplex Red oxidation will be measured in the presence of
           glutamate (10 mmol/L), malate (2 mmol/L), and succinate (10 mmol/L). The fluorescent
           signal will be corrected for background auto-oxidation and calibrated to a standard
           curve. The H2O2 production rates will be expressed relative to mitochondrial protein.

        -  Third, simultaneous assessments of the oxygen consumption and carbon dioxide production
           will be determined through blood gas measurements from the arteriovenous samples
           obtained from the splanchnic and skeletal muscle tissue beds. These assessments will be
           performed at all three time points of blood sample assessments and correlated with the
           measured TCA cycle flux in their respective tissue beds.

        -  Circulating exosomes will also be derived from the arteriovenous samples of the
           splanchnic and skeletal muscle tissue beds to determine its intra-exosome proteome and
           metabolome and its relationship with the TCA cycle flux in their respective tissue beds.
           Incorporation of D5-phenylalanine will help trace the protein formation in the exosomes.

        -  Finally, changes in the whole body metabolome and proteome determined via the
           arteriovenous samples obtained from the splanchnic and skeletal muscle tissue beds will
           also be performed and correlated with the TCA cycle flux in their respective tissue
           beds.
    

Intervention name(s): Somatostatin; Glucagon
Intervention type(s): Drug; Drug
Intervention description(s): Somatostatin infusion to create a low insulin state.; Glucagon infusion in the setting of ongoing somatostatin.
Other intervention name(s): growth hormone-inhibiting hormone; Glucagen
Intervention MeSH terms: Glucagon; Hormones; Somatostatin
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: TCA cycle flux; in vivo measurements; Stable isotopes; Arteriovenous balance; Metabolome; Proteome; Exosomes