Monday Morning Start time: 07:30am
A. 7:30 Registration
B. 8:30 Welcome (Dr. Maren Laughlin,
senior advisor / NIDDK, NIH)
C. 8:40 Principles of Metabolic flux and Use of Radioactive
Isotopes (O. McGuinness)
Learning Objectives > (1) What is metabolic flux and how can tracer dilution principles
be used to quantify flux? (2) Responsible
Conduct of Research in the use of radioactive isotopes. (3) How
does one optimize the measurement of radioactivity of compounds labeled with 14C,
3H or 32P? (4) How does one measure a metabolic
rate using 13C or 3H tracers? (5) What
are the difficulties and limitations of the use of radioactive isotopes to
measure metabolic rates?
Sections > (A) Basic Principles of Metabolic flux (define tracer methodology
and principle of isotope dilution). (B) Measurement of beta radioactivity by scintillation
counting (Conversion of cpm to dpm (external standards, automatic
quench correction, internal standards); How does one deal with counting
artifacts (quenching, chemiluminescence)).
(C) Principles of measurement of metabolic rates (Notion of
specific activity of labeled precursor; Problems and solutions with variations
of specific activity of precursor (how does one avoid dealing with one equation
and two variables). (D) Limitations of the use of isotopes for
metabolic studies (Difference between transfer of label and net flux;
Isotopic exchanges; Isotopic equilibration without or with ATP consumption).
D. 9:50 break
E. 10:10-10:45 Problem Breakout> Four numerical problems of increasing complexity are presented to the
attendees to teach them how to plan real-life experiments with radioactive
isotopes without guessing the amount of radioactivity to be used. The attendees are given ~30 min to find the
solutions. Then, the instructor and the attendees discuss how they went about
working on the problems.
F. 10:45-11:30 Problem Discussion
Start time: 01:30pm
A. 1:30-3:00 Basic Concepts in Mass
Spectrometry (R. Wolfe)
Learning Objectives > (1) gain an understanding of the main mass spectrometry techniques
used to investigate metabolic processes with stable isotopes. (2)
Become familiar with current expressions of isotopic enrichment, including
Tracer:Tracee Ratio and atom (or
mol) percent excess. (3) Learn how to measure isotopic
enrichment by mass spectrometry (basic approaches). (4)
Learn how to calculate isotopic enrichment using Gas Chromatography-Mass
Spectrometry and LC-MS/MS.
Sections > (A) Basic
Description of Instrumentation: Isotope
ratio mass spectrometry (IRMS); Gas Chromatography-Mass Spectrometry (GC-MS); Gas
Chromatography-Combustion-Isotope Ratio Mass Spectrometry (GC-C-IRMS); Liquid
Chromatography-Mass Spectrometry (LC-MS; LC-MS/MS).
(B) Calculation of Enrichment with IRMS: Correction of enrichment for background enrichment
– Tracer:Tracee Ratio (TTR) vs. Molar Percent Enrichment (MPE); skew correction factor to
correct for the fact that the natural distribution of mass isotopomers is the
same in the sample and the background; d)
Use of a standard to calculate enrichment; measurement of 13C-enrichment
after combustion; effect of sample size on observed ratio. (C)
Calculation of Enrichment with GC-MS (definition of total ion chromatogram, mass
spectrum, and selected ion monitoring (SIM); identifying appropriate
fragment(s) to monitor; calculation of theoretical abundance; calculation of
isotopic enrichment using SIM; effect of skewed abundance of tracer, skew
correction factor; overlapping spectra correction, calculation of TTR when
TTR> 1 (using multiple ions to calculate isotopic enrichment, using less
abundant masses to measure low levels of enrichment, calculation of
concentration by internal standard technique).
B. 3:00-3:15 Break
C. 3:15-3:50 Homework
> Students are asked to calculate isotopic
enrichment from GC-MS data
D. 3:50-4:20 Problem Discussion
“Free time to explore Nashville”
Start time: 09:00am
A. 9:00-10:30 Measurement Of Metabolic Fluxes With Isotopic
Tracers (R. Wolfe)
Learning Objectives > (1) Responsible
Conduct of Research in human and animal investigations.
(2) Gain a conceptual and practical
understanding of calculating the rate of substrate appearance (Ra) by tracer
dilution using a single pool model with radioactive and stable isotopes. (3)
Understand the benefit of priming the substrate pool, how to calculate a
tracer priming dose, and the limitations of the primed-constant infusion
technique. (4) Understand the basic approach for calculating substrate
oxidation using a metabolic tracer. (5) Understand the calculation of
fractional synthetic rate.
Sections > (A) Tracer
Kinetics-Single Pool Models (Constant
infusion of tracer; Influence of changes in uptake on calculation of rate of
appearance; Calculation of Ra with a bolus injection of tracer; Priming the
pool; Estimation of Ra in the non-steady state; Minimizing errors by curve
fitting). (B) Incorporation Studies (Principles and calculation of substrate
oxidation at the whole body level using tracers, including use of Atom Percent
Excess vs. Tracer:Tracee Ratio;
Bicarbonate recovery factor; Improving the estimation of true precursor
enrichment; Priming the bicarbonate pool; Determination of carbon dioxide
production with labeled bicarbonate; Problems in determining oxidation with
tracers; Labeled CO2 reincorporation; Contribution of naturally
occurring 13C to apparent CO2 enrichment; Fractional
synthetic rate; Synthetic rate). (C) Non
steady-state kinetics. (Single and multiple pool models).
B. 10:30-10:45 Break
C. 10:45-11:45 Glucose Kinetics / including the euglycemic
clamp (O. McGuinness)
> (1) Responsible Conduct of
Research in such types of investigations in rodents and humans. (2) Define
the physiological correlates of glucose flux. (3) Learn best practices
for experimental design optimization and data interpretation to evaluate
Sections > (A)
Overview of Glucose Kinetics (Define
steady state; Define the relationship between glucose concentration and glucose
mass in the body; Identify sites and relative rates of glucose production and
consumption and how these rates differ among species). (B) What
Are The Sources of Glucose Appearance? (Understand what 'production' is, from a tissue
point of view; define the relative contribution of the liver and kidney to
glucose production). (C)
How Do We Get Started? (Choosing a
tracer; understand how the sites of sampling and infusion can influence the
measured rates of glucose flux; know how to optimize the study design to
maximize steady state conditions). (D)
Assessing Insulin Action (Choosing a
tracer; Know how fasting status influences insulin action differently in mice
and humans; Define what insulin action is in the liver and the periphery;
Understand what a euglycemic hyperinsulinemic clamp is and how to deal with
variable rates of endogenous insulin and glucagon secretion; How to recognize
and deal with tracer/model assumption errors (non steady-state and negative
endogenous production rates); Be able to evaluate data used to calculate
hepatic and peripheral insulin action; Understand the principles used in
assessing tissue specific glucose uptake).
A. 1:30-2:30 Assessing glucose flux and insulin action using isotopic
tracers in the Human (M. Cree
Objectives > (1)
Understand how to translate a physiologic hypothesis that insulin action is
altered to a tracer study to quantify the site and magnitude of the defect. (2) Understand the experimental
protocol(s) followed to evaluate insulin action 3) Understand how to control
for patient population variables that impact outcomes.
Sections > (A) Why is glucose homeostasis altered? (Insulin
action vs Insulin secretion vs insulin independent glucose action. (B) Glucose tolerance and insulin secretion:
oral vs intravenous glucose delivery. (C) The experimental protocol to quantify insulin action: Hyperinsulinemic
euglycemic clamp and limb clamps. (C) Consideration
of disease and environment in the design. (Patient health/disease state,
Pre-study preparation: Diet, exercise, medications, fasting/fed states).
B. 2:30-2:45 Break
C. 2:45-4:00 Lipid Metabolism: Basic Kinetics (E. Parks)
LEARNING OBJECTIVES > (1) To understand the principles and limitations of various types
of measurements of lipid metabolism using stable isotopes. (2) Recognize that
glycerol and fatty acid availability are very sensitive to insulin and other
hormones. Fatty acid oxidation and triglyceride metabolism in
SECTIONS > (A) Lipolysis and Fatty Acid Release: Their flux rates can be assessed
using glycerol fatty acid tracer as well as substrate cycling between
triglycerides and fatty acids. (B) Fatty
Acid Oxidation (Pathways of fatty acid oxidation; Citric acid cycle
exchange reactions; in vivo assessment of CPT activity). (C) Multiple substrate pools contribute to
lipoprotein and intracellular triglyceride synthesis; limitations of various
methods for measuring intracellular lipid synthesis.
Start time: 07:00pm
A. Introduction to the NIH Grants Process (M. Laughlin)
B. Insulin and
a. Application to animal models (O. McGuinness)
b. Application to human models (M. Cree-Green and R. Wolfe)
Start time: 08:30am
Measurements of Energy Expenditure (S.
Learning Objectives > (1) Outline different methods for quantifying energy expenditure
(or CO2 production) . (2) Identify the pros/cons for
each. (3) Outline the general principle of using “doubly labeled water”,
listing important criteria for the experimentalist. (4) Explain the rationale for different data
Sections > (A) Overview
of energy expenditure - Where does “energy” go? How Do I Quantify Tissue-Specific Rates of CO2
Production? a) Arterio-venous balance is required. b) Single vs. multiple compartments. c) Concerns about mixing/complete
perfusion. How do I quantify substrate-specific
rates of CO2 production?
a) Measure the production of 13C-labeled CO2. b) Concerns
about the recovery of a labeled substrate.
do i quantify total body CO2 production? a) Direct
calorimetry. b) Indirect calorimetry (Direct measurements of gas exchange;
indirect measurements of gas exchange (i.e.:
“doubly-labeled” water)). How Do I
Process the Data and Normalize the Results? Note that all the MMPC centers are currently involved in a NIDDK study
on defining rules for standardizing rates of energy expenditure (measured by
indirect calorimetry) in C57BL mice of different ages and weights across
centers. Analysis of covariance with learning modules is freely available on
the MMPC website for all centers to compare their data.
B. 10:00-10:15 Break
10:15-11:45 Measure Synthesis of Proteins, Fats, Sterols,
Glucose & Nucleic Acids with 2H2O (S.
Learning Objectives > (1) General equations for calculating rates of synthesis in
short-term vs. long-term studies, i.e. those that run over several hours vs. those that run over several days,
respectively. (2) Why 2H2O is a unique tracer for measuring
the synthesis of various macromolecules.
(3) Explain why one requires
knowledge of the labeling of specific hydrogen(s) in a product molecule to
accurately determine its rate of synthesis.
(4) Contrast the pros/cons of
using GC-MS vs. NMR to measure the
labeling of molecules.
Sections > What can be done with 2H2O that cannot be done with other tracers? a)
Simultaneous tracing of multiple processes. Choice between acute and chronic labeling studies? a) Source(s) of blood glucose (acute). b)
Total triglyceride dynamics (acute and chronic). c) Protein synthesis _
acute and chronic: (Single vs.
multiple proteins; ii. 2H2O vs. H218O). Complementary Approach to Glucose-Insulin Clamping: a) Measurements
of flux during metabolic steady state vs.
Start time: 01:30pm
1:30-2:00 Measuring Synthesis of Adenine Nucleotides, Coenzyme A, Nucleic Acids (Brunengraber) Learning Objectives > (1) Identify problems associated with the use of isotopic tracers
for very long experiments (weeks or months).
(2) Long-term isotopic
experiments occur in an open biological system where unlabeled foodstuffs enter
the system continuously. (3) During long-term isotopic
experiments, salvaged pathways recycle labeled intermediates into de novo synthesis pathways.
2:00 -2:45 Practical
applications of Physiological Models using Stable Isotopes I (Kelleher)
Learning objectives: (1)
To understand methods for describing isotopes in physiological studies. (2)
To learn a practical method for solving for isotopic mixtures. (3)
To understand the role of experimental error in developing and testing models. (4) To understand the different methods
for solving for rates of synthesis and their limitations.
Sections Topics: Describing
stable isotope tracers. Solving for tracer contribution to mixtures with simple
linear regression. Introduction to Pre-steady state labeling. Solving for the
rate of synthesis using nonlinear regression
in Protein Metabolism (Wolfe)
Learning Objectives > (1) Understand how to use of whole body protein turnover
techniques. (2) Earn how to calculate the rate of synthesis of individual
proteins. (3) Learn how to measure tissue protein and amino acid kinetics
using tracers and transorgan balance techniques.
Sections > Whole body protein turnover: a) Catabolic and anabolic states. b) Energy cost of protein synthesis. c) Stochastic model of whole body protein turnover. d) Comparison of tracers; Isotopic determination of urea production. e) Single amino acid tracer kinetics to calculate whole body
protein turnover. Measurement of Protein FSR: a) Constant tracer infusion. b) Flooding dose tracer injection. c) Sub-flooding dose tracer injection. Methods to Estimate Precursor Enrichment for
Measurement of FSR: a) Fractional breakdown rate. b) Constant tracer infusion. c) Bolus injection. Arterio-Venous
Model: a) Measurement of A-V balance. b) 3-pool
and 4-pool models of protein kinetics and amino acid transport. c) Measurement
of tissue oxidation rate. d) Technical aspects of performing A-V
balance studies from mouse to human.
Start time: 07:00pm
Social Hour / Dinner At Wild Horse saloon
Thursday Morning Start time: 08:30am
of Positional Isotopomer Analysis to Assess Pathway Fluxes (G. Cline, M. Merritt, D.
Learning Objectives > (1) Understand the basic principles of NMR. (2) Understand
how the information content of NMR data differs from MS data. (3) Understand
how metabolic flux information is extracted from NMR data. (4) Review
common applications of NMR to metabolic flux measurements.
A. 8:30-9:15 NMR in Tracer Metabolism /
Merritt: Basic NMR Principles (Measurement of
fractional enrichment, spin-spin coupling, multiplet analysis; measuring 13C
and 2H isotopomer distribution).
B. 9:15-10:00 Applications to Biochemical Physiology:
Steady State Measurements of Metabolic Fluxes (Cline): Metabolic pathways in isolated cells (TCA cycle, anaplerosis,
and substrate cycling); Calculating hepatic fluxes by multinuclear NMR
(glycogen synthesis pathways, gluconeogenesis and glycogenolysis, TCA cycle
Applications: Kinetic Analysis of Metabolic Fluxes/
Befroy - Practical
aspects of performing in vivo
experiments (homogeneity, localization, lipid suppression etc.). b) Conventional
13C labeling strategies (Brain / Muscle). c) Alternative
13C labeling strategies (Brain / Liver). d) Complementary in vivo techniques
E. 11:10-12:00 Evolving techniques
(Merritt): a) Hyperpolarization-intracellular
Start time: 01:30pm
Use of Mass Isotopomer Distribution Analysis (Kelleher,
Learning Objectives > (1) To appreciate the multiple uses of mass isotopomer distribution
for metabolic investigation, with the understanding that mass isotopomer
distributions and positional isotopomer distributions yields complementary
insights on metabolic regulation.
Applications (Puchowicz) a) Measurement of low analyte
enrichment by oligomerization of analyte. b)
Use of hexamethylenetetramine to amplify the 2H-enrichment on
glucose carbons, which can be converted to formaldehyde; measurement of low 2H-
or 18O-enrichment of water. c) Measurement of low 2H-enrichment
of analytes by isotope fractionation.
B. 2:00-3:15 Practical applications of
Physiological Models using Stable Isotopes II (Kelleher): Learning objectives: (1) To understand key
differences in using stable and radioisotopes. (2) To understand the
difference between linear and non-linear models. (3) To understand the
complexities of isotope incorporation studies. (4)To develop strategies for
identifying and dealing with underdetermined models. Sections: Stable and radioisotopes, which to choose
Linear versus nonlinear models, superposition Nonlinear Model for lipid
synthesis from 13C precursors. What to do if the model does not fit
the data? Overdetermined and underdetermined
C. 3:15-3;30 Break
D. 3:30-5:00 Optional computer workshops
a. NMR Workshop (D. Befroy) > Dynamic
modeling analysis of NMR spectroscopy data. Free downloadable MatLab software
will be available.
b. Metabolic Flux Analysis Workshop (J. Young)> Metabolic Flux Analysis
using the MFA Suite of tools with GC-MS data.
Investigations of pathway regulation + pathway discovery
(metabolomics associated with mass isotopomer distribution).
Start time: 07:00pm
Trainees Presentations (10)
8:30-9:30 PATHWAY DISCOVERY THROUGH
METABOLOMICS ASSOCIATED WITH STABLE ISOTOPE TECHNOLOGIES (h.
Learning Objectives > Limitations of
non-targeted metabolomics, used as a single research tool, to investigate the
regulation of metabolic pathways. Changes in relative concentrations do not
reflect changes in flux rates. The association
of metabolomics and stable isotope technology allows to follow C, H, N of
substrates through the metabolome. This leads to the identification of new
pathways and new regulatory mechanisms. Metabolomics should be integrated with
classical tools used to investigate metabolism: flux rates, enzyme
activity/regulation, balance studies
9:30-10:30 Inherently Difficult Problems (H. Brunengraber).
Learning Objectives > (1) Appreciate limitations on the use of isotopes for metabolic
studies, using examples of problems, which have challenged investigators for
many years. (2) Measurement of Cori cycling with labeled lactate. (3) Measurement
of fatty acid oxidation in vivo. (4) Measurement
of glucose production across a high blood flow organ (kidney, intestine). (5) Glyceroneogenesis. (6)
Ketogenesis vs. pseudoketogenesis. (7) Measurement of coenzyme A and
nucleic acid turnover with 2H-enriched water. (8) Impacts
of secondary tracers on the process investigated (e.g., (i) formation of [13C]ketone
bodies from infused [13C]fatty acids), and (ii) formation of [13C]glucose
from infused [13C]propionate). (9)
Impact of loads of labeled substrates on metabolic processes being traced.
Lunch Boxes will be available for your trip home.