novel stable isotope methods to assess metabolic fluxes using microscale...
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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 [email protected]
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Fluxes provide information about system bottlenecks and regulation
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Metabolic flux changes cannot be inferred from enzyme expression
Burgess et al., Cell Metabolism 5, 313–320, 2007.
Glucose
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Static metabolite abundances are not reliable indicators of flux
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How to determine metabolic fluxes?
Start by measuring external fluxes
Solve for internal fluxes using mass balances
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But external measurements usually aren’t enough
Lactate
Glucose
Glycerol
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But external measurements usually aren’t enough
Lactate
Glucose
Glycerol
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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
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Metabolic flux analysis uses math models to decipher labeling data
Wiechert Metab Eng 3:195-206, 2001
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Math model comprises mass balances and isotopomer balances on each intracellular metabolite
Wiechert Metab Eng 3:195-206, 2001
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Fluxes are regressed by fitting the model to match the labeling data
Adjustfluxes
a
b
c
d
e
Simulated Experimental
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Fluxes are regressed by fitting the model to match the labeling data
Simulated Experimental
Adjustfluxes
a
b
c
d
e
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Fluxes are regressed by fitting the model to match the labeling data
Adjustfluxes
a
b
c
d
e
Simulated Experimental
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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
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Comparison of CHO flux maps
EarlyExponential Decline
TCA TCA TCA TCA
LateExponential Stationary
Templeton et al. 2013. Biotech and Bioeng 110:2013-24.
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Treatment of airway epithelial cells with cigarette smoke condensate
Rahman et al. JCI Insight 1:e88814, 2016.
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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)
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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.
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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.
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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.
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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.
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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
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Stable isotope methods are needed to examine intermediary fluxes in vivo
EndoRa
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Combined 2H and 13C tracers have been used to assess liver CAC and GNG fluxes
a
b
c
d
e
Glycerol
Lactate
AA, FAGlycogen
[U-13C3]-propionate
2H2O
2H2O2H2O
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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
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GC-MS approach for measuring stable isotope enrichment of glucose
GC-MSanalysis
Antoniewicz et al. Anal Chem 83:3211-6, 2011.
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Vein Artery
Sample
Erythrocytes
2H2O +[6,6-2H2]Glucose
[U-13C3]Propionate
Dual catheter system enables continuous infusion and sampling in conscious mice
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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
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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
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Short- and long-term fasted mice are differentially enriched at steady state
Hasenour et al., Am. J. Physiol. Endocrinol. Metab. 309:E191, 2015.
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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.
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Model tracks carbon and hydrogen atom transitions
a
b
c
d
e
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
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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)
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Method captures expected changes between short and long-term fasting
Hasenour et al., Am. J. Physiol. Endocrinol. Metab. 309:E191, 2015.
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Method captures expected changes between short and long-term fasting
→ EndoRa
a
b
c
d
e
Glycerol
Lactate
AA, FAGlycogen
Glucose
short n=5, long n=7; VEndoRa p=0.25
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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
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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
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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
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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
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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
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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
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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