Novel stable isotope methods to assess metabolic fluxes using microscale samples

Jamey D. YoungAssociate Professor and Chancellor’s Faculty FellowChemical and Biomolecular EngineeringMolecular Physiology and BiophysicsVanderbilt Universityj.d.young@vanderbilt.edu

Fluxes provide information about system bottlenecks and regulation

Metabolic flux changes cannot be inferred from enzyme expression

Burgess et al., Cell Metabolism 5, 313–320, 2007.

Glucose

Static metabolite abundances are not reliable indicators of flux

How to determine metabolic fluxes?

Start by measuring external fluxes

Solve for internal fluxes using mass balances

But external measurements usually aren’t enough

Lactate

Glucose

Glycerol

But external measurements usually aren’t enough

Lactate

Glucose

Glycerol

Isotope tags allow us to “measure” intracellular fluxes

M0 M1 M2 M3

Abu

ndan

ceMID measured by GC- or LC-MS

Pathway 4Pathway 1

Pathway 3

+

Pathway 2

+13C12C

MID = Mass Isotopomer Distribution

Metabolic flux analysis uses math models to decipher labeling data

Wiechert Metab Eng 3:195-206, 2001

Math model comprises mass balances and isotopomer balances on each intracellular metabolite

Wiechert Metab Eng 3:195-206, 2001

Fluxes are regressed by fitting the model to match the labeling data

Adjustfluxes

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Simulated Experimental

Fluxes are regressed by fitting the model to match the labeling data

Simulated Experimental

Adjustfluxes

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b

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d

e

Fluxes are regressed by fitting the model to match the labeling data

Adjustfluxes

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d

e

Simulated Experimental

INCA: Isotopomer Network Compartmental Analysis

Custom MFA software

Handles transient and steady-state labeling experiments

Generalizable to any metabolic network of interest

Recently adapted to MS/MS and NMR isotopomer measurements

mfa.vueinnovations.comYoung Bioinformatics 30:1333-1335, 2014

Comparison of CHO flux maps

EarlyExponential Decline

TCA TCA TCA TCA

LateExponential Stationary

Templeton et al. 2013. Biotech and Bioeng 110:2013-24.

Treatment of airway epithelial cells with cigarette smoke condensate

Rahman et al. JCI Insight 1:e88814, 2016.

Pancreatic islets sense glucose and secrete insulin to control glycemia

Levine and Leibowitz, Towards gene therapy of diabetes mellitus, Mol. Med., 1999

G6PC2

Glucose stimulated insulin secretion (GSIS)

Quantifying glucose cycling in mouse pancreatic islets

Novel stable isotope analyses demonstrate significant rates of glucose cycling in mouse pancreatic islets. Wall ML, Pound LD, Trenary I, O'Brien RM, Young JD. Diabetes 64:2129-37, 2015.

GC-MS approach for measuring positional isotope enrichment of glucose

Measuring deuterium enrichment of glucose hydrogen atoms by gas chromatography/mass spectrometry. Antoniewicz MR, Kelleher JK, Stephanopoulos G. Anal Chem 83:3211-6, 2011.

Islets from chow fed mice incubated in 5mM D7-glucose

Novel stable isotope analyses demonstrate significant rates of glucose cycling in mouse pancreatic islets. Wall ML, Pound LD, Trenary I, O'Brien RM, Young JD. Diabetes 64:2129-37, 2015.

Islets from chow fed mice incubated in 11mM D7-glucose

Novel stable isotope analyses demonstrate significant rates of glucose cycling in mouse pancreatic islets. Wall ML, Pound LD, Trenary I, O'Brien RM, Young JD. Diabetes 64:2129-37, 2015.

SummaryGlucose Cycling (%)

Diet [Glucose] (mM) WT G6pc2

KOChow 5 16 ± 4 2 ± 2High fat 5 25 ± 3 0.5 ± 2Chow 11 40 ± 6 3 ± 1High fat 11 35 ± 5 3 ± 1

• Rates of glucose cycling in WT islets greater than reported by prior methods

• Islets from G6pc2 KO mice exhibit negligible cycling rates compared to WT islets under all conditions

• Glucose cycling has averaged ~10% in preliminary studies of donor human islets

Stable isotope methods are needed to examine intermediary fluxes in vivo

EndoRa

Combined 2H and 13C tracers have been used to assess liver CAC and GNG fluxes

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b

c

d

e

Glycerol

Lactate

AA, FAGlycogen

[U-13C3]-propionate

2H2O

2H2O2H2O

NMR analysis of glucose positional enrichment is the gold standard but is difficult to scale down to the mouse

Jones et al. Am J Physiol Endocrinol Metab, 2001

2H NMR spectra 13C NMR spectra of C-2β

GC-MS approach for measuring stable isotope enrichment of glucose

GC-MSanalysis

Antoniewicz et al. Anal Chem 83:3211-6, 2011.

Vein Artery

Sample

Erythrocytes

2H2O +[6,6-2H2]Glucose

[U-13C3]Propionate

Dual catheter system enables continuous infusion and sampling in conscious mice

Short-term (9h) vs. long-term (19h) fasting study

Overall objective: Test and validate a scaled-down, low-cost, high-throughput GC-MS-based in vivo flux analysis approach

Hypothesis: GC-MS analysis of glucose 2H/13C enrichment will yield sufficient information to precisely assess GNG and CAC fluxes

Isotopic steady state is reached within 90 min of isotope infusion

Hasenour et al., Am. J. Physiol. Endocrinol. Metab. 309:E191, 2015.

short (n=5) long (n=7) m/z 301

Short- and long-term fasted mice are differentially enriched at steady state

Hasenour et al., Am. J. Physiol. Endocrinol. Metab. 309:E191, 2015.

An isotopomer model was developed to simulate the movement of tracers through liver metabolism

Hasenour et al., Am. J. Physiol. Endocrinol. Metab. 309:E191, 2015.

Model tracks carbon and hydrogen atom transitions

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Lactate

AA, FA

Lac (ABbCcde) → Pyr (ABCcde) + H (b)

O

O

O

O

OH O

Lactate

Lactate Dehydrogenase

Pyruvate

NAD+

NADH

A Bb Ccde A B Ccde

INCA ModelID Equation

GLCinf Gluc.inf (AaBbCcDdEeFfg) → Gluc.ext (AaBbCcDdEeFfg)

Hinf H.inf (a) → H (a)

Hsink H → Sink

PCC PropCoA (ABabCcde) + CO2 (D) → SuccCoA (ACcdBabD) + H (e)

SDH SucCoA (ABabCcdD) + H (e) + H (f) → 0.5*Oac (ABCefD) + 0.5*Oac (DCBefA) + H (a) + H (b) + H (c) + H (d)

CS Oac (ABCcdD) + AcCoA (EFfgh) → Cit (DCcdBFfgEA) + H (h)

IDH Cit (ABabCDcdEF) + H (e) → Akg (ABCeaDcdE) + H (b) + CO2 (F)

OGDH Akg (ABCabDcdE) → SucCoA (BCabDcdE) + CO2 (A)

PDH Pyr (ABCabc) → AcCoA (BCabc) + CO2 (A)

GPI F6P (AabBCcDdEeFfg) + H (h) → G6P (AbBhCcDdEeFfg) + H (a)

ALDO DHAP (CchBAab) + GAP (DdEeFfg) → F6P (AabBCcDdEeFfg) + H (h)

GAPDH BPG (ABbCcd) + H (e) + H (f) → 0.5*GAP (AfBeCcd) + 0.5*DHAP (AefBCcd) + H (b)

ENO PEP (ABCcd) + H (b) → BPG (ABbCcd)

PK PEP (ABCab) + H (c) → Pyr (ABCabc)

PC Pyr (ABCcde) + CO2 (D) + H (f) + H (g) → 0.5*Oac (ABCfgD) + 0.5*Oac (DCBfgA) + H (c) + H (d) + H (e)

PCK Oac (ABCabD) → PEP (ABCab) + CO2 (D)

PYGL Glycogen (AaBbCcDdEeFfg) + H (h) → G6P (AaBhCcDdEeFfg) + H (b)

MPI F6P (AabBCcDdEeFfg) + H (h) → F6P (AahBCcDdEeFfg) + H (b)

GK Glycerol (AaeBbCcd) + H (f) → 0.5*DHAP (AfeBCcd) + 0.5*GAP (AeBfCcd) + H (a) + H (b)

LDH Lac (ABbCcde) → Pyr (ABCcde) + H (b)

Method captures expected changes between short and long-term fasting

Hasenour et al., Am. J. Physiol. Endocrinol. Metab. 309:E191, 2015.

Method captures expected changes between short and long-term fasting

→ EndoRa

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b

c

d

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Glycerol

Lactate

AA, FAGlycogen

Glucose

short n=5, long n=7; VEndoRa p=0.25

Method captures expected changes between short and long-term fasting

→ Glycogen

a

b

c

d

e

Glycerol

Lactate

AA, FAGlycogen

short n=5, long n=7; VPYGL p=2E-04

Method captures expected changes between short and long-term fasting

→ Glycerol

a

b

c

d

e

Glycerol

Lactate

AA, FAGlycogen

short n=5, long n=7; VGK p=0.025

Method captures expected changes between short and long-term fasting

→ CAC

a

b

c

d

e

Glycerol

Lactate

AA, FAGlycogen

short n=5, long n=7; VCAC p=0.049

Relative fluxes have similar values and precision to NMR-based results

Contribution to EndoRa

This study (n=7)

Satapati et al.*(n=6-8)

Glycogen 0.005 ± 0.004 0.02 ± 0.02Glycerol 0.256 ± 0.008 0.28 ± 0.02PEP 0.739 ± 0.009 0.70 ± 0.02

*Satapati et al. J Lipid Res, 2012

Flux relative to CAC

This study (n=7)

Satapati et al.*(n=6-8)

Enolase 1.59 ± 0.09 1.7 ± 0.1 Pyruvate cycling 1.44 ± 0.08 2.8 ± 0.1PEPCK 3.03 ± 0.14 4.5 ± 0.2

Future studies and applications

Ongoing validation studies: different tracers, additional plasma/tissue measurements

Establish a Metabolic Flux Analysis (MFA) Subcore within the Vanderbilt MMPC Will provide stable isotope analysis of tracer

studies involving mice and mouse tissues Apply method to study metabolic

pathophysiology in vivo L-AMPK WT vs KO comparison Effects of FFA on liver metabolism Exercise studies

Summary 2H/13C flux analysis can be used to dissect

metabolic physiology in cells and in vivo We have developed a novel GC-MS-based

2H/13C MFA approach that can be applied to examine mouse liver metabolism in vivo Requires only 40 µL plasma Mice are conscious and unrestrained Higher throughput and lower cost than NMR

INCA software enables isotopomer models to be quickly modified to test assumptions or to incorporate new tracers, measurements, pathways

AcknowledgementsCollaborators O’Brien lab – G6PC2 Wasserman lab – in vivo MFAYoung lab Martha Wall Clint Hasenour Irina TrenaryFunding NIH T32 (Martha, Clint) MMPC MICROMouse Program Vanderbilt Discovery Award NIH R01 DK106348 Martha

Clint

Wasserman

R. O’Brien