In Vivo Assessment of Cellular Metabolism in Humans

Study Overview
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Brief Summary
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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
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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.

Official Title
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In Vivo Assessment of the Tricarboxylic Acid Cycle Flux in the Muscle and Splanchnic Bed of Humans: A Pilot Study

Conditions
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Normal Cellular Metabolism

Intervention / Treatment
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* Drug: Somatostatin
* Drug: Glucagon


Participation Criteria
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Eligibility Criteria
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INCLUSION CRITERIA: Ages 18-45 Able to provide written consent EXCLUSION CRITERIA: Diabetes mellitus or impaired fasting glucose levels (fasting blood glucose >110mg/dl). Renal Failure Pregnancy Steroid use Muscle Disease Liver Disease Major Depression Anemia H/O alcohol use Medications other than OCPs BMI of 30 or greater

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 45 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| No Intervention: Control Group<br>No somatostatin and glucagon infusions | |
| Active Comparator: Intervention Group<br>Somatostatin and glucagon infusions | Drug: Somatostatin<br>* Somatostatin infusion to create a low insulin state.<br>* Other names: growth hormone-inhibiting hormone;Drug: Glucagon<br>* Glucagon infusion in the setting of ongoing somatostatin.<br>* Other names: Glucagen;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| In Vivo TCA cycle flux in skeletal muscle and splanchnic tissue | Normal healthy study participants will receive an initial priming dose followed by a continuous infusion of 2-13C-Acetate, 2-15N-Glutamine and D5-Phenylalanine in order to achieve steady state enrichment of 13C and 15N in their system. Serial arteriovenous blood samples will be obtained from the femoral artery, femoral vein and hepatic vein and serial skeletal muscle tissue biopsies will be obtained from the vastus lateralis. These samples will be analyzed by GC-MS and NMR spectroscopy to quantify the isotopomer intermediates of the TCA cycle and measure the corresponding TCA cycle flux. The flux estimations from the arteriovenous blood samples will be compared to that obtained directly from the skeletal muscle tissue. This methodology will be validated in the setting of low insulin levels alone or in combination with high glucagon concentrations. | 12 hours | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in the protein and metabolite contents within circulating exosomes derived from the arterial-venous blood supply of the skeletal muscle and splanchnic tissue |  | 12 hours | 
| Changes in the metabolome derived from the arterial-venous blood supply of the skeletal muscle and splanchnic tissue |  | 12 hours | 
| Changes in the proteome derived from the arterial-venous blood supply of the skeletal muscle and splanchnic tissue in response to hormonal manipulation. |  | 12 hours | 
| Oxygen consumption in skeletal muscle and splanchnic tissue in response to hormonal manipulation |  | 12 hours | 
| Mitochondrial respiration in skeletal muscle tissue |  | 12 hours | 
| Reactive oxygen species emissions in skeletal muscle tissue |  | 12 hours | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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TCA cycle flux, in vivo measurements, Stable isotopes, Arteriovenous balance, Metabolome, Proteome, Exosomes