Agilent Technologies, Inc. · NYSE:A

Agilent Technologies — Technical Presentation (July 2017)

54 slides from Agilent Technologies's technical presentation, Jul 20, 2017

  1. Slide 1: Combining Seahorse XF analysis with stable isotope tracing to reveal novel drug targets

    Combining Seahorse XF analysis with stable isotope tracing to reveal novel drug targets for metabolic and neurodegenerative disease Ajit Divakaruni, Ph.D. July 20, 2017 Asst. Professor of Molecular & Medical Pharmacology, UCLA For Research Use Only. Not for use in diagnostic procedures.

  2. Slide 2: Thiazolidinediones (TZDs)

    Thiazolidinediones (TZDs) Rosiglitazone (Avandia®) Pioglitazone (Actos®) Effective insulin sensitizers with prohibitive side effects Is PPARγ agonism the only mechanism of action? For Research Use Only. Not for use in diagnostic procedures.

  3. Slide 3: Thiazolidinediones (TZDs)

    Thiazolidinediones (TZDs) Rosiglitazone (Avandia®) Pioglitazone (Actos®) Effective insulin sensitizers with prohibitive side effects Is PPARγ agonism the only mechanism of action? TZDs bind to mitochondrial membranes at Rapid in vitro and in vivo effects clinically relevant EC50 ≈ 1μM concentrations

  4. Slide 4: Metabolism

    Metabolism For Research Use Only. Not for use in diagnostic procedures. © 2016 F. Hoffmann-La Roche Ltd

  5. Slide 5: A "black box" approach

    A “black box” approach SUGARS/AMINO ACIDS/ (1) MEASURE RATES OF FATTY ACIDS INPUTS AND OUTPUTS (Respiration, lactate efflux) Quantitative flux through lactate an intact network Real-time rates upon acute perturbations CO2+H2O O2 consumed For Research Use Only. Not for use in diagnostic procedures.

  6. Slide 6: A "black box" approach

    A “black box” approach SUGARS/AMINO ACIDS/ (1) MEASURE RATES OF FATTY ACIDS INPUTS AND OUTPUTS (Respiration, lactate efflux) specific inhibitors Quantitative flux through lactate an intact network adjust rate control Real-time rates upon acute perturbations CO2+H2O O2 consumed For Research Use Only.

  7. Slide 7: Respiration is a powerful experimental tool to study mitochondrial metabolism

    Respiration is a powerful experimental tool to study mitochondrial metabolism For Research Use Only. Not for use in diagnostic procedures. Adapted from Divakaruni and Brand (2011) Physiology

  8. Slide 8: Respiration can measure any process that makes or consumes ATP

    Respiration is a powerful experimental tool because it can measure any process that either makes or consumes ATP For Research Use Only. Not for use in diagnostic procedures.

  9. Slide 9: Is there a direct effect of TZDs on mitochondrial function?

    Is there a direct effect of TZDs on mitochondrial function? RESPIRATION IN CULTURED MYOCYTES Pioglitazone (Actos®) For Research Use Only. Not for use in diagnostic procedures. see Divakaruni…and Jastroch (2014) Meth. Enzym.

  10. Slide 10: Is there a direct effect of TZDs on mitochondrial function?

    Is there a direct effect of TZDs on mitochondrial function? RESPIRATION IN CULTURED MYOCYTES Force mitochondria to respire independently of cellular ATP demand H+ H+ H+ H+ H+ H+ H+ H+ H+ H+ FCCP H+ IMS H+ ATP Synthase Q"cycle Inner'Membrane Matrix IV H+ II III H+ I O2 H O H+ 2 NADH H+ NAD + H+ H+ H+

  11. Slide 11: Pioglitazone has a direct, acute effect on mitochondrial function

    Pioglitazone has a direct, acute effect on mitochondrial function For Research Use Only. Not for use in diagnostic procedures. see Divakaruni…and Jastroch (2014) Meth. Enzym.

  12. Slide 12: Decreased respiratory capacity could be due to dozens of possibilities

    Decreased respiratory capacity could be due to dozens of possibilities 400 FCCP Oxygen consumption rate Control rotenone/ (pmol O 2/min/min) Pioglitazone antimycin A 300 oligomycin 200 100 0 0 16 32 48 Time (min) For Research Use Only. Not for use in diagnostic procedures.

  13. Slide 13: Permeabilized cells provide control over mitochondrial substrate provision

    Permeabilized cells provide control over mitochondrial substrate provision Intact cell Permeabilized cell From Fiskum G...and Lehninger AL (1980) PNAS Validation in Divakaruni...and Murphy (2013) PNAS; Protocols in Divakaruni…and Murphy (2014) Curr. Prot. Tox. Image from Fiskum…and Lehninger (1980)

  14. Slide 14: TZDs acutely inhibit mitochondrial pyruvate uptake

    TZDs acutely inhibit mitochondrial pyruvate uptake From Divakaruni...and For Research Use Only. Not for use in diagnostic procedures. Murphy (2013) PNAS

  15. Slide 15: The mitochondrial pyruvate carrier (MPC)

    The mitochondrial pyruvate carrier (MPC) • MPC Knockdown lowers TZD EC50 • UK5099 reproduces plasma membrane glucose uptake and AMPK phosphorylation Divakaruni and Murphy (2013) PNAS • 125I-pioglitazone binds to MPC Colca and Kletzein (2013) PLoS ONE For Research Use Only. Not for use in diagnostic

  16. Slide 16: The mitochondrial pyruvate carrier (MPC)

    The mitochondrial pyruvate carrier (MPC) • MPC Knockdown lowers TZD EC50 • UK5099 reproduces plasma membrane glucose uptake and AMPK phosphorylation Divakaruni and Murphy (2013) PNAS • 125I-pioglitazone binds to MPC Colca and Kletzein (2013) PLoS ONE A METABOLOMICS APPROACH? (1) Metabolite changes a

  17. Slide 17: Using stable knockdown to understand the cellular response to reduced MPC activity

    Using stable knockdown to understand the cellular response to reduced MPC activity KNOCKING DOWN MPC COMPONENTS IN MYOCYTES DOESN’T ALTER MITOCHONDRIAL ATP PRODUCTION (!) For Research Use Only. Not for use in diagnostic procedures. From Vacanti, Divakaruni...and Metallo (2014) Mol. Cell

  18. Slide 18: Using stable knockdown to understand the cellular response to reduced MPC activity

    Using stable knockdown to understand the cellular response to reduced MPC activity KNOCKING DOWN MPC COMPONENTS IN MYOCYTES DOESN’T ALTER MITOCHONDRIAL ATP PRODUCTION (!) For Research Use Only. Not for use in diagnostic procedures. From Vacanti, Divakaruni...and Metallo (2014) Mol. Cell

  19. Slide 19: Metabolism, two ways

    Metabolism, two ways (1) MEASURE RATES OF (2) TAKE A DETAILED INPUTS AND OUTPUTS SNAPSHOT IN TIME (Metabolomics, stable isotope tracing) Granular detail from metabolite abundances Relative enrichment of intermediates from labeled substrates For Research Use Only. Not for use in diagnostic procedures

  20. Slide 20: Combining respiration and stable isotope tracing to fully reveal cell metabolism

    Combining respiration and stable isotope tracing to fully reveal cell metabolism Isotopically labeled substrates (i.e. glucose, glutamine, etc.) are offered as tracers 13C labeling of metabolite pools can map the pattern of carbon flux For Research Use Only. Not for use in diagnostic procedures.

  21. Slide 21: Combining respiration and stable isotope tracing to fully reveal cell metabolism

    Combining respiration and stable isotope tracing to fully reveal cell metabolism Isotopically labeled substrates (i.e. glucose, glutamine, etc.) are offered as tracers 13C labeling of metabolite pools can map the pattern of carbon flux For Research Use Only. Not for use in diagnostic procedures.

  22. Slide 22: The MPC is a switch that can potentiate oxidation of amino acids and fatty acids

    The MPC is a switch that can potentiate the oxidation of amino acids and fatty acids MYOCYTES ADAPT TO DECREASED MITOCHONDRIAL PYRUVATE UPTAKE BY: 1. Increasing glutamine oxidation 2. Increasing palmitate oxidation For Research Use Only. Not for use in diagnostic procedures.

  23. Slide 23: Stable isotope tracing can reveal information that respirometry cannot

    Stable isotope tracing can reveal information that respirometry cannot For research use only. Not for use in diagnostic procedures. For Research Use Only. Not for use in diagnostic procedures.

  24. Slide 24: Malic enzyme activity increases upon MPC knockdown

    Malic enzyme activity increases upon MPC knockdown For Research Use Only. Not for use in diagnostic procedures.

  25. Slide 25: The MPC is a "switch" that can potentiate metabolic flexibility

    The MPC is a “switch” that can potentiate metabolic flexibility MYOCYTES ADAPT TO DECREASED MITOCHONDRIAL PYRUVATE UPTAKE BY: 1. Increasing amino acid oxidation 2. Increasing palmitate oxidation 3. Increasing malic enzyme flux For Research Use Only. Not for use in diagnostic procedures. Adapted from

  26. Slide 26: The MPC is a "switch" that can potentiate metabolic flexibility

    The MPC is a “switch” that can potentiate metabolic flexibility Metabolic flexibility is a characteristic of healthy skeletal muscle Adapted from Kelley DE (2005) JCI For Research Use Only. Not for use in diagnostic procedures.

  27. Slide 27: The MPC is a "switch" that can potentiate metabolic flexibility

    The MPC is a “switch” that can potentiate metabolic flexibility MPC inhibition potentiates fatty acid oxidation (by reducing malonyl CoA?) An inability to increase lipid oxidation when faced with high supply can cause increased muscle fat accumulation and insulin resistance Adapted from Hue and Taeg

  28. Slide 28: Is there a metabolic component to neurodegenerative disease?

    Is there a metabolic component to neurodegenerative disease? TYPE 2 DIABETES IS A STRONG RISK FACTOR FOR AD SHARED PATHOLOGY BETWEEN T2D AND AD • Insulin resistance • Cognitive impairment • Amyloidosis • Neural tissue atrophy • Oxidative stress Healthy brain Mild-to-moderate AD From Small GW…and Phe

  29. Slide 29: Is there a metabolic component to neurodegenerative disease?

    Is there a metabolic component to neurodegenerative disease? TYPE 2 DIABETES IS A STRONG RISK FACTOR FOR AD SHARED PATHOLOGY BETWEEN T2D AND AD • Insulin resistance • Cognitive impairment • Amyloidosis • Neural tissue atrophy • Oxidative stress REPURPOSING OF ANTI-DIABETIC THERAPIES FOR AD • Intrana

  30. Slide 30: Viability is unchanged despite reduced MPC activity

    Viability is unchanged despite reduced MPC activity For Research Use Only. Not for use in diagnostic procedures. Divakaruni…and Murphy (2017) J. Cell Biol.

  31. Slide 31: Global bioenergetics unchanged despite reduced MPC activity

    Global bioenergetics unchanged despite reduced MPC activity For Research Use Only. Not for use in diagnostic procedures.

  32. Slide 32: Global bioenergetics unchanged despite reduced MPC activity

    Global bioenergetics unchanged despite reduced MPC activity For Research Use Only. Not for use in diagnostic procedures.

  33. Slide 33: Challenging neurons by stimulating glutamate receptors

    Challenging neurons by stimulating glutamate receptors Glutamate is the dominant excitatory neurotransmitter in the brain (balanced by inhibitory GABA) For Research Use Only. Not for use in diagnostic procedures.

  34. Slide 34: Challenging neurons by stimulating glutamate receptors

    Challenging neurons by stimulating glutamate receptors Glutamatergic neurotransmission comes at a tremendous energetic cost For Research Use Only. Not for use in diagnostic procedures.

  35. Slide 35: Challenging neurons by stimulating glutamate receptors

    Challenging neurons by stimulating glutamate receptors Under conditions of energetic stress or de-energization (stroke, epilepsy, some chronic neurodegeneration), extracellular glutamate rises to toxic levels For Research Use Only. Not for use in diagnostic procedures.

  36. Slide 36: MPC inhibition protects from excitotoxic cell death

    MPC inhibition protects from excitotoxic cell death (!) In rich culture medium, inhibiting mitochondrial pyruvate metabolism in neurons protects from excitotoxic neuronal death For Research Use Only. Not for use in diagnostic procedures.

  37. Slide 37: MPC inhibition protects from excitotoxic cell death

    MPC inhibition protects from excitotoxic cell death (!) In rich culture medium, inhibiting mitochondrial pyruvate metabolism in neurons protects from excitotoxic neuronal death How has MPC inhibition rewired neuronal metabolism in a way that is neuroprotective? For Research Use Only. Not for use in

  38. Slide 38: Neurons have a huge capacity to oxidize ketone bodies and BCAAs

    Bioenergetic analysis shows neurons have a huge capacity to oxidize ketone bodies and BCAAs… Acute challenge in simple salts medium where substrate preference is assumed a priori by the experimenter For Research Use Only. Not for use in diagnostic procedures.

  39. Slide 39: Stable isotope tracing shows no increase in oxidation of these substrates

    …but stable isotope tracing shows no increase in oxidation of these substrates in response to UK5099 In rich culture medium we see no increase in TCA enrichment from ketone bodies (left, pink) or leucine (right, green) upon MPC inhibition over 24 hr. For Research Use Only. Not for use in diagnostic

  40. Slide 40: Stable isotope tracing shows no increase in oxidation of these substrates

    …but stable isotope tracing shows no increase in oxidation of these substrates in response to UK5099 WE MISSED SOMETHING For Research Use Only. Not for use in diagnostic procedures.

  41. Slide 41: Searching for the protective mechanism of UK5099

    Searching for the protective mechanism of UK5099 Amino acid levels suggest increased neuronal glutamate oxidation upon reduced pyruvate uptake For Research Use Only. Not for use in diagnostic procedures.

  42. Slide 42: Searching for the protective mechanism of UK5099

    Searching for the protective mechanism of UK5099 Glutamate is generally not considered a metabolic substrate due to its central (and presumably compartmentalized) role in neurotransmission For Research Use Only. Not for use in diagnostic procedures.

  43. Slide 43: UK5099 selectively increases glutamate oxidation

    UK5099 selectively increases glutamate oxidation Stable isotope tracing reveals increased glutamine/glutamate oxidation in cells and organotypic slices cortical neurons For Research Use Only. Not for use in diagnostic procedures.

  44. Slide 44: UK5099 selectively increases glutamate oxidation

    UK5099 selectively increases glutamate oxidation Stable isotope tracing reveals increased glutamine/glutamate oxidation in cells and organotypic slices cortical neurons organotypic hippocampal slices For Research Use Only. Not for use in diagnostic procedures.

  45. Slide 45: Anaplerosis may explain selective glutamate oxidation upon MPC inhibition

    Anaplerosis may explain why glutamate oxidation is selectively increased upon MPC inhibition UK5099 For Research Use Only. Not for use in diagnostic procedures.

  46. Slide 46: Anaplerosis may explain selective glutamate oxidation upon MPC inhibition

    Anaplerosis may explain why glutamate oxidation is selectively increased upon MPC inhibition A 3-13C1 glucose tracer can be used to measure CO2-fixing reactions from glucose that replenish the TCA cycle For Research Use Only. Not for use in diagnostic procedures.

  47. Slide 47: Anaplerosis may explain selective glutamate oxidation upon MPC inhibition

    Anaplerosis may explain why glutamate oxidation is selectively increased upon MPC inhibition A 3-13C1 glucose tracer can be used to measure CO2-fixing reactions from glucose that replenish the TCA cycle For Research Use Only. Not for use in diagnostic procedures.

  48. Slide 48: The glutamate mobilized for oxidation is not discrete from the evoked release pool

    The glutamate mobilized for oxidation is not discrete from the pool apportioned for evoked release Glutamate released by depolarization from veratridine (prevents Na+-channel closure) and ouabain (Na+/K+-ATPase inhibitor) For Research Use Only. Not for use in diagnostic procedures.

  49. Slide 49: Can attenuated vesicular release explain the protection from excitotoxic injury?

    Can attenuated vesicular release explain the protection from excitotoxic injury? Limiting depolarization-induced glutamate release could minimize the positive-feedback, excitotoxic cascade For Research Use Only. Not for use in diagnostic procedures.

  50. Slide 50: Can attenuated vesicular release explain the protection from excitotoxic injury?

    Can attenuated vesicular release explain the protection from excitotoxic injury? Depolarization induced with a concentration & duration of NMDA that does not compromise viability For Research Use Only. Not for use in diagnostic procedures.

  51. Slide 51: Pyruvate carrier inhibition lowers synaptic glutamate release

    Pyruvate carrier inhibition lowers synaptic glutamate release UK5099 attenuates excitatory post-synaptic currents in primary mouse pyramidal neurons For Research Use Only. Not for use in diagnostic procedures.

  52. Slide 52: Our model

    Our model Divakaruni…and Murphy (2017) J. Cell Biol. For Research Use Only. Not for use in diagnostic procedures.

  53. Slide 53: Our model

    Our model Divakaruni…and Murphy (2017) J. Cell Biol. For Research Use Only. Not for use in diagnostic procedures.

  54. Slide 54: Acknowledgements

    Anne&N.&Murphy,&Ph.D. AcknowledgementsDavid&A.&Ferrick,&Ph.D. Theodore'P.'Ciaraldi,'Ph.D. Robert'R.'Henry,'M.D. George'W.'Rogers,'Ph.D. Melvin'I.'Simon,'Ph.D. Aleksander'Y.'Andreyev,'Ph.D. Sandra'E.'Wiley,'M.S. Susanna'Petrosyan,'B.S. Anne Murphy ChrisFan'M.'Metallo,'Ph.D. Christian Metallo Morton'P

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