In Vivo Assessment of Cellular Metabolism in Humans

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

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.

Study Overview

• Status: Completed
• Conditions: Normal Cellular Metabolism
• Intervention / Treatment: Drug: Somatostatin; Drug: Glucagon

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.

• Study Type: Interventional
• Enrollment (Actual): 17
• Phase: Phase 1

Contacts and Locations

Study Locations

• United States
  • Minnesota
    • Rochester, Minnesota, United States, 55905
      • Mayo Clinic in Rochester

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 45 years (ADULT)
• Accepts Healthy Volunteers: Yes
• Genders Eligible for Study: All

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: BASIC_SCIENCE
• Allocation: RANDOMIZED
• Interventional Model: PARALLEL
• Masking: NONE

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
NO_INTERVENTION: Control Group; No somatostatin and glucagon infusions
ACTIVE_COMPARATOR: Intervention Group; Somatostatin and glucagon infusions	Drug: Somatostatin; Somatostatin infusion to create a low insulin state.; Other Names: growth hormone-inhibiting hormone; Drug: Glucagon; Glucagon infusion in the setting of ongoing somatostatin.; Other Names: Glucagen

What is the study measuring?

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	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

Collaborators and Investigators

• Sponsor: K. Sreekumaran Nair
• Collaborators: National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

Publications and helpful links

General Publications

• Jones JG, Solomon MA, Cole SM, Sherry AD, Malloy CR. An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans. Am J Physiol Endocrinol Metab. 2001 Oct;281(4):E848-56. doi: 10.1152/ajpendo.2001.281.4.E848.
• Akram M. Citric acid cycle and role of its intermediates in metabolism. Cell Biochem Biophys. 2014 Apr;68(3):475-8. doi: 10.1007/s12013-013-9750-1.
• Befroy DE, Perry RJ, Jain N, Dufour S, Cline GW, Trimmer JK, Brosnan J, Rothman DL, Petersen KF, Shulman GI. Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy. Nat Med. 2014 Jan;20(1):98-102. doi: 10.1038/nm.3415. Epub 2013 Dec 8.
• Sunny NE, Parks EJ, Browning JD, Burgess SC. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab. 2011 Dec 7;14(6):804-10. doi: 10.1016/j.cmet.2011.11.004.
• Schumann WC, Magnusson I, Chandramouli V, Kumaran K, Wahren J, Landau BR. Metabolism of [2-14C]acetate and its use in assessing hepatic Krebs cycle activity and gluconeogenesis. J Biol Chem. 1991 Apr 15;266(11):6985-90.
• Alves TC, Pongratz RL, Zhao X, Yarborough O, Sereda S, Shirihai O, Cline GW, Mason G, Kibbey RG. Integrated, Step-Wise, Mass-Isotopomeric Flux Analysis of the TCA Cycle. Cell Metab. 2015 Nov 3;22(5):936-47. doi: 10.1016/j.cmet.2015.08.021. Epub 2015 Sep 24.

Helpful Links

• Mayo Clinic Clinical Trials

Study record dates

Study Major Dates

• Study Start: July 1, 2016
• Primary Completion (ACTUAL): March 2, 2017
• Study Completion (ACTUAL): March 2, 2017

Study Registration Dates

• First Submitted: April 12, 2016
• First Submitted That Met QC Criteria: April 19, 2016
• First Posted (ESTIMATE): April 22, 2016

Study Record Updates

• Last Update Posted (ACTUAL): August 3, 2022
• Last Update Submitted That Met QC Criteria: August 2, 2022
• Last Verified: August 1, 2022

More Information

Terms related to this study

• Keywords: Stable isotopes; Metabolome; Exosomes; Proteome; TCA cycle flux; in vivo measurements; Arteriovenous balance
• Additional Relevant MeSH Terms: Physiological Effects of Drugs; Gastrointestinal Agents; Hormones, Hormone Substitutes, and Hormone Antagonists; Glucagon; Hormones; Somatostatin
• Other Study ID Numbers: 16-000085; U24DK100469 (NIH)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: Yes
• Studies a U.S. FDA-regulated device product: No