1. Redox States and Phosphorylation Potentials Bob Harris raharris@iupui.edu October 5, 2010

2. Redox States NAD + /NADH cytoplasm NAD + /NADH mitochondria NADP + /NADPH cytoplasm NADP + /NADPH mitochondria

3. Measuring the NAD + redox state Usually expressed as ratio of [NAD +] /[NADH] Total NAD + divided by total NADH? Free NAD + divided by free NADH? Make any difference if we use total values or free values?

4. Cytosolic NAD + /NADH ratios based on total (free and bound) NAD + and NADH in rat liver StateNAD + NADH NAD + /NADH  mol/g Fed0.760.145.4 Starved0.820.165.1

5. Cytosolic NAD + /NADH ratios based on free concentrations StateNAD + /NADH Fed 725 Starved 528

6. Calculating the NAD + redox state Free values obtained by measuring metabolites of an equilibrium enzyme Lactate + NAD + pyruvate + NADH + H + K eq = [pyruvate][NADH][H + ] / [lactate][NAD + ] [NAD + ]/[NADH] = [pyruvate][H + ] / [lactate] x 1/K eq

7. Equilibrium constants Equilbrium constants: for A B; K eq = [B]/[A] Mass action ratios: MAR = [B]/[A] Equilibrium enzymes:high activity; K eq = MAR Nonequilibrium enzymes:low activity; K eq = MAR A B C D E F

8. Cytoplasmic free NAD + /NADH Lactate dehydrogenase catalyzes equilibrium reaction: Lactate + NAD + pyruvate + NADH + H + K eq = [pyruvate][NADH][H + ] / [lactate][NAD + ] [NAD + ]/[NADH] = [pyruvate][H + ] / [lactate] x K eq Set pH = 7.0 and incorporate into K eq K’ eq = [pyruvate][NADH]/ [lactate][NAD + ] [NAD + ]/[NADH] = [pyruvate]/[lactate] x 1/K’ eq

9. Example of calculation Freeze clamp liver of fed wild type mice: Lactate: 1.09  0.09  mol/g wet wt Pyruvate: 0.12  0.01  mol/g wet wt K’ eq @ pH 7.0 = 1.11 x 10 -4 [NAD + ]/[NADH] = [pyruvate]/[lactate] x 1/K’ eq [NAD + ]/[NADH] = [0.120]/[1.09] x 1/1.11 x 10 -4 [NAD + ]/[NADH] = 991

10. Effect of ethanol on liver cytosolic NAD + /NADH ratio Ethanol + NAD + acetaldehyde + NADH + H + Expect NADH drive pyruvate to lactate via: Pyruvate + NADH +H + Lactate + NAD + Expect decrease in NAD+/NADH ratio

11. Effect of ethanol on liver cytosolic NAD/NADH ratio TreatmentNAD/NADH Control719 Ethanol (2 millimoles) 132* * Five minutes after injection of ethanol.

12. Equilibrium enzymes used for calculations of free ratios Mitochondrial free NAD + /NADH:  -hydroxybutyrate dehydrogenase  -hydroxybutyrate + NAD + acetoacetate + NADH + H + K’ eq @ pH 7.0 = 4.93 x 10 -2 Glutamate dehydrogenase Glutamate + NAD + yields  -ketoglutarate + NADH + NH4 + K’eq @ pH 7.0 = 3.87 x 10 -3 mM

13. Effect of starvation on liver mitochondrial NAD + redox state StateNAD + /NADHNAD + /NADH (Free)* (Total) Fed 7.3 2.2 Starved 4.7 ND *Calculated from concentrations of components of the glutamate dehydrogenase reaction.

14. Effect of ethanol on liver mitochondrial NAD/NADH ratio Ethanol + NAD + acetaldehyde + NADH + H + Acetaldehyde + NAD + acetate + NADH + H + Expect NADH will drive  -ketoglutarate to glutamate via:  -Ketoglutarate + NADH +NH 4 + glutamate + NAD + Expect decrease in mitochondrial NAD + /NADH ratio

15. Effect of ethanol on liver mitochondrial NAD + /NADH ratio TreatmentNAD + /NADH Control7.7 Ethanol (2 millimoles) 2.7* * Five minutes after injection of ethanol.

16. Equilibrium enzymes used for calculations of free ratios Cytoplasmic free NADP + /NADPH 6-phosphogluconate dehydrogenase: 6-phosphogluconate + NADP + ribulose 5- phosphate + NADPH + H + + CO 2 Isocitrate dehydrogenase: Isocitrate + NADP +  -ketoglutarate + NADPH + CO 2 Malic enzyme: Malate + NADP + pyruvate + NADPH + H + + CO 2

17. K eq for NADP + coupled enzymes 6-phosphogluconate dehydrogenase 1.17 M Isocitrate dehydrogenase 1.72 x 10 -1 M Malic enzyme 3.44 x 10 -2 M

18. Reactions catalyzed by NADP + coupled enzymes produce CO 2 CO 2 concentration does not vary significantly under conditions that are normally studied. Rather than measure, usually assumed to be 1.16 mM. Caution: CO 2 concentration is affected by changes in pH.

19. Typical values of cytoplasmic NADP + /NADPH StateNADP + /NADPH NADPH/NADP + Fed 0.009110 Starved 0.006175

20. NADP + /NADPH ratio important Sets the ratio of GSH/GSSG in cytoplasm because of equilibrium enzyme reaction catalyzed by glutathione reductase NADPH + H + +GSSG 2 GSH + NADP + Driven far to the right because of very high NADPH/NADP + ratio. Important in both cytoplasm and mitochondrial matrix space

21. NAD + /NADH ratio important for many reasons High cytoplasmic NAD/NADH ratio favors oxidation of substrates. Low cytoplasmic NAD/NADH results in low pyruvate and low oxaloacetate which inhibits glucose synthesis. Free NAD + is activator of SIRT1 Free NADH is activator of the PDKs Both serve as both substrates and allosteric effectors for many enzyme systems.

22. Phosphorylation potential Defined as [ATP]/[ADP][Pi] Comes from:∆G = ∆Gº - RTln[ATP]/[ADP][Pi] Two ways of determining –From measurements of total ATP, ADP, and Pi (not accurate because total [ADP] >>free [ADP]) –From concentrations of metabolites of equilibrium enzymes (much more accurate)

23. Calculation of phosphorylation potentials [ATP]/[ADP][Pi] = [NAD + ]/[NADH] x [glyceraldehyde-3-P]/[3-phosphoglycerate] x K GAPDH x K PGK

24. Derivation Glyceraldehyde-3-P + NAD + + Pi yields 1,3- bis-Phosphoglycerate + NADH + H + 1,3-Phosphoglycerate + ADP yields 3-Phosphoglycerate + ATP Sum: Glyceraldehyde-3-P + NAD + + Pi + ADP yields 3- phosphoglycerate + ATP + NADH K GAPDH x K 3-PGK = [ATP]/[ADP][Pi] x [NADH]/[NAD + ] x 3- [phosphoglycerate]/ [glyceraldehyde-3-P] [ATP]/[ADP][Pi] = [NAD + ]/[NADH] x [glyceraldehyde-3-P]/[3- phosphoglycerate] x K GAPDH x K 3-PGK

25. Calculation of phosphorylation potentials [ATP]/[ADP][Pi] = [NAD + ]/[NADH][H + ] x [glyceraldehyde- 3-P]/[3-phosphoglycerate] x K GAPDH x K 3-PGK Obtain [NAD +] /[NADH] from [pyruvate]/[lactate] and K LDH [ATP]/[ADP][Pi] = [pyruvate]/[lactate] x [glyceraldehyde- 3-P]/[3-phosphoglycerate] x {K GAPDH x K 3-PGK }/K LDH

26. Calculation of phosphorylation potentials Obtain [glyceraldehyde-3-P] from [dihydroxyacetone-P] and the K eq (22) for triose phosphate isomerase glyceraldehyde-3-P dihydroxyacetone-P K eq = 22 = [dihydroxyacetone-P]/ [glyceraldehyde-3-P] [glyceraldehyde-3-P] = [dihydroxyacetone-P]/22

27. Calculation of phosphorylation potentials [ATP]/[ADP][Pi] = [pyruvate]/[lactate] x [dihydroxyacetone phosphate]/22 x 1/[3- phosphoglycerate] x {K GAPDH x K 3-PGK }/K LDH {K GAPDH x K 3-PGK }/K LDH = 1.65 x 10 7 M -1

28. Typical metabolite values for freeze clamped rat liver Metabolite  mol/g wet wt Lactate 1.36 Pyruvate0.258 3-Phosphglycerate0.387 Dihydroxyacetone P0.043 ATP3.38 ADP1.32 AMP0.294 P i 4.76

29. Calculation of phosphorylation potentials ATP ADP ATP/ADPxP i *  mol/g wet wtM -1 Total 3.38 1.32 531 Free 16,300 *[P i ] taken to be 4.8  mol/g

30. Calculation of free [ADP] Free cytosolic [ADP] = [ATP]/{[Pi] x phosphorylation potential}

31. Calculation of phosphorylation potentials ATP ADPATP/ADPxP i *  mol/gM -1 Total 3.38 1.32 531 Free 3.38 0.046 16,300 *[P i ] taken to be 4.8  mol/g; water content taken to be 0.8 grams per gram wet weight tissue.

32. Calculation of free [AMP] From the equilibrium constant (1.05) for reaction catalyzed by myokinase: ATP + AMP 2 ADP Free cytosolic [AMP] = {[free cytosolic ADP] 2 x K MK }/[measured ATP]

33. Comparison of total measured [AMP] and calculated free [AMP] ADPAMP  mol/g wet wt Total 1.320.294 Free cytosolic 0.0460.0007* *0.7 nmoles/g wet weight!

34. Important points about adenine nucleotides Free [AMP] is much lower than total [AMP] - Important because [AMP] activates AMPK and functions as positive or negative allosteric effector for many enzymes. Free [ADP] is much lower than the total [ADP] –Important because [ADP] determines respiration rate of mitochondria Decrease in [ATP] results in increase in [AMP] because of equilibrium reaction catalyzed by myokinase: 2 ADP ATP + AMP

35. Effect of fasting, exercise, hypoglycemia, high fat diet, and diabetes on liver adenine nucleotides Berglund et al. JCI 119:2412–2422 (2009)

36. Hems and Brosnan. Effect of ischemia on content of metabolites in rat liver and kidney Biochem J 1970; 120:105-111 Well-fed rats IschemiaATPADPAMP AMP/ATP (sec) (  mol/g wet wt) 02.71.30.26 0.09 601.61.80.85 0.53 48-starved rats IschemiaATPADPAMP AMP/ATP (sec) (  mol/g wet wt) 01.72.00.64 0.37 600.91.71.65 1.83

37. Greenbaum et al. Hepatic metabolites and …. in animals of different dietary and hormonal status. Arch. Biochem. Biophys. 1971; 143: 617-663 Metabolic State ATPADPAMP AMP/ATP (  mol/g wet wt) Well-fed 1.90.910.23 0.12 Starved1.71.00.31 0.18

38. Schewenke et al. Mitochondrial and cytosolic AT/ADP ratios in rat liver in vivo Biochem J 1981; 200: 405-408 Metabolic State ATPADPAMP AMP/ATP (  mol/g dry wt) Well-fed 3.30.860.16 0.05 Starved2.70.820.18 0.07

39. Perhaps mice are not just small rats?

40. Our measurements on fed and fasted mice Measurement Fed Fasted  mol/g wet wt ATP 3.0  0.2 3.2  0.2 ADP 0.89  0.07 0.85  0.07 AMP 0.28  0.04 0.24  0.03

41. Why difference between our data and the data of Burgess et al.? Freeze clamping has to be done rapidly to preserve phosphorylation state of the adenine nucleotides. Burgess et al. Approximately 20 seconds. Our study: Less than 8 seconds.

42. Faupel et al. The problem of tissue sampling from experimental animals….. ABB 1972; 148: 509-522

43. Faupel et al. The problem of tissue sampling from experimen -tal animals…. ABB 1972; 148: 509- 522

44. Freeze clamp protocol 1.Three people who can work together are required. One to manage stop watch; one strong person to handle freeze clamps; one person with good hands to kill mouse by cervical dislocation, open mouse with a single cut with scissors, tear out liver, and place on freeze clamp. 2.Practice until steps 4, 5, and 6 can be completed by team in less than 8 seconds. Discard any samples not clamped in less than 8 seconds. 3. Handle mice on several days prior to the experiment in the room in which the mice will be killed. Transport the mice to the killing room one at a time. 4. Person 1: start stopwatch at time of cervical dislocation; stop at time liver clamped. 5. Person 2: kill mouse by cervical dislocation with large pair of scissors; open mouse with a single cut with same scissors; tear out liver by hand; place liver on freeze clamps. 6. Person 3: clamp tissue with as much force as possible with liquid- nitrogen cooled clamps. 7. Clean the area and instruments before bringing the next mouse to the killing room. (Mice are stressed by the odor of blood).

45. Effect of fasting, exercise, hypoglycemia, high fat diet, and diabetes on liver adenine nucleotides Berglund et al. JCI 119:2412–2422 (2009)

46. Our measurements on chow and high fat fed mice Measurement Chow High Fat Diet  mol/g wet wt ATP 3.0  0.2 2.7  0.2 ADP 0.89  0.07 1.14  0.05* AMP 0.28  0.04 0.42  0.02* *P < 0.05

47. Best way to measure ATP, ADP, and AMP? Enzyme-coupled assays? HPLC?

48. Direct comparison of enzymatic and HPLC method for nucleotide quantification Measurement Enzymatic HPLC  mol/g wet wt ATP 3.0  0.2 2.7  0.2 ADP 0.89  0.07 1.8  0.1* AMP 0.28  0.04 0.6  0.1* *P < 0.05

49. References Faupel, RP, Seitz, HJ, Tarnowski, W., Thiermann, V, Weiss, C. The problem of tissue sampling from experimental animals with respect to freezing technique, anoxia, stress and narcosis. ABB (1972) 148: 509-522. Veech, RL, Guynn, R, Veloso, D. The time-course of the effects of ethanol on the redox and phosphorylation states of rat liver. Biochem. J. (1972) 127, 387-397. Veech, RL, Lawson, JWR, Cornell, NW, Krebs, HA. Cytosolic phosphorylation potential. JBC (1979) 254: 6538-6547. Berglund,ED, Lee-Young, RS, Lustig, DG, Lynes, SE, Donahue,P, Camacho, RC., Meredith, ME., Magnuson, MA, Charron, MJ, Wasserman, DH. Hepatic energy state is regulated by glucagon receptor signaling in mice. JCI (2009) 119: 2412-2422.

51. Importance of AMP/ATP ratio AMP is a positive allosteric effector of: –Glycogen phosphorylase (glycogenolysis) –PFK1 (glycolysis) –AMP kinase (glycolysis; Fatty acid oxidation; inhibit gluconeogenesis) ATP is a negative allosteric effector of: –Pyruvate kinase (glycolysis)

54. Resveratrol High fat diet SIRT1 SREBP1c PGC1  FOXFAS Ethanol PPAR 

55. Lipoic acid

56. Shong et al. The effect of feeding high fat diet on NQO1 expression. In preparation

57. Park et al. Lipoic Acid Decreases Lipogenesis via AMPK-Dependent and –Independent Pathways. Hepatology 2008; 48:1477-1486 Lipoic acid in diet –reduced hepatic steatosis. –increased AMPK activity –Inhibited SREBP1c expression –Increased capacity for fatty acid oxidation

58. Park et al. Lipoic Acid Decreases Hepatic Lipogenesis Through AMPK-Dependent and AMPK-Independent Pathways HEPATOLOGY, Vol. 48, No. 5, 2008

63. Park et al. Lipoic Acid Decreases Hepatic Lipogenesis Through AMPK-Dependent and AMPK-Independent Pathways HEPATOLOGY, Vol. 48, No. 5, 2008

64. NQO1 NQO1 = “old yellow enzyme” = DT diaphorase (D = DPN (NAD + ) ; T = TPN (NADP + ); NAD(P)H:quinone acceptor oxidoreductase; cytoplasmic enzyme NAD(P)H + H + + electron acceptor (EA) yields NAD(P) + + H 2 EA –Important point: catalyzes 2 electron transfer as opposed to one electron transfer that could produce O 2

65.  -Lapachone Best known synthetic substrate for NQO1 – Lowest Km; highest Vmax NAD(P)H + H + +  Lap yields NAD(P) + +  LapH 2 Approved in some countries as anti- cancer agent

66. Effect of  -Lapachone in fat-fed mice Hwang et al. Stimulation of NADH oxidation ameliorates obesity and related phenotypes in mice. Diabetes 58: 965-974, 2009 Increased hepatic NAD + /NADH ratio. –Increased AMPK activity –Increased PGC1  and SIRT1 – Decreased acetyl-CoA carboxylase activity –Increased fatty acid oxidation –Ameliorated adiposity, glucose intolerance, dyslipidemia, and fatty liver in mice fed high fat diet

67. Shin et al.  -Lapachone alleviates alcoholic fatty liver disease in rats. In preparation In alcohol-fed mice,  -Lapachone –reduced hepatic steatosis –Increased hepatic fatty acid oxidizing capacity –Increases NAD/NADH ratio –Increased AMPK activity

68. High fat diet Resveratrol (PDK KO???) SIRT1 SREBP1c PGC1  FOXFAS Ethanol NAD + Smile ERR  PDK4 p53 PDK2

70. Phenotype of NQO1 knockout mice Decreased hepatic NAD/NADH ratio Reduces fasting blood levels of glucose in chow fed and high fat fed mice Reduces steatosis in high fat fed mice