Role of the Pyruvate

Transcription

1 Role of the Pyruvate Dehydrogenase Complex in the Regulation of Blood Glucose Robert A. Harris Indiana University School of Medicine Indianapolis, Indiana Kyungpook National University School of Medicine Daegu, Korea

2 BCAAs (Leu, Ile, Val) AAs Glucose FAs Lactate t Pyruvate FA-CoAs Cytosol BCAAs Pyruvate Mitosol NAD + FA-CoAs α-ketoglutarate CO 2 PDC + Glutamate NADH BCKAsNAD Acetyl-CoA CO 2 BCKDC Oxaloacetate Citrate NADH Acyl-CoAs TCA Cycle AAs Glutamate AAs Succinyl-CoA CO 2 α-ketoglutarate NADH KGDC NAD+

3 PDC OP ADP Inactive H 2 O (-) (-) (-) (-) PDK 1,2,3,4 (+) PDH P ase 1 PDH P ase 2 ATP Pi Produced from glucose CoASH Pyruvate NAD + PDC OH Active CO 2 Acetyl-CoA NADH Produced by fatty acid oxidation

4 Pyruvate Dehydrogenase Kinases Four isoforms (PDK1,2,3,4) that are expressed in a tissue specific manner Encoded by separate nuclear genes All four inducible

5 Pyruvate Dehydrogenase Phosphatases Two isoforms (PDP1 and 2) that are expressed in tissue specific manner Encoded by separate nuclear genes Both inducible

6 PDH Complex Structuret E2/E3BP Inner Core Domain PRP PDP E3BP E3 Domain E1 Binding Domain β β α α E Lipoyl Domain E2 Lipoyl Domain 2 PDK PDK Site 1 Site 2 E2 Lipoyl Domain 1 E3 Site 3

7 Core of 48 E2 and 12 E3BP subunits each with a swinging arm bearing 2 lipoyl groups Rate-limiting enzyme 10 to 30 E1 α 2 β 2 heterotetramers E1α phosphorylation blocks binding of the lipoyl group of the E2 swinging arm swinging arm 6 E3 homodimers Variable number of PDK homodimers attached to the lipoyl groups of the swinging arms of E2

8 Some things we have learned about inhibition of PDC by the PDKs Phosphorylation of site 1 alone inactivates PDC. All four PDKs phosphorylate sites 1 and 2. Only PDK1 phosphorylates site 3. Phospho serine specific anitibodies are commercially available for all three sites. PDKs are recruited to PDC by binding to the inner lipoyl domain of the E2 swinging arm.

9 Some things we have learned about inhibition of PDC by the PDKs Reduction and acetylation of the lipoyl group of E2 stimulates PDK1, PDK2, and PDK4 activity but not PDK3 activity. This explains stimulatory effects of NADH and acetyl- CoA on PDK activity and why fatty acid oxidation inhibits PDC activity. Pyruvate inhibits PDK activity by binding in a small pocket in the N-terminal domains of PDK1, PDK2, and PDK4.

10 Starvation increases expression of PDK4 in Tissues of the Mouse Muscle Heart Kidney Liver PDK K4 (Arbitra ary units) P < Fed Starved P < Fed Starved P < Fed Starved 4 P < Fed Starved

11 Factors that regulate PDK and PDP expression PDC OP ADP (+) Inactive Hypoxia H 2 O Insulin (-) (-) ATP PDK1 PDK2 PDK3 PDK4 (+) (+) (+) Starvation/ Diabetes Fatty acids/ glucocorticoids (-) PDP1 PDP2 Pi CoASH Pyruvate NAD + PDC OH Active CO 2 Acetyl-CoA NADH

12 Transcription factors that regulate PDK expression PDC OP ADP Inactive H 2 O PPARα/δ; FOXO; GR; ERRα/γ; HNF4α; STAT5 PDK1 PDK2 PDK3 PDK4 HIF-1α PDP1 PDP2 ATP Pi CoASH Pyruvate NAD + PDC OH Active CO 2 Acetyl-CoA NADH

13 The Pyruvate Dehydrogenase Complex is inactivated during Fasting PDC is inactivated because: PDK activity is stimulated by NADH and acetyl- CoA produced by fatty acid oxidation PDK activity is increased by up regulation of PDK4 and PDK2 Purpose: Conserves three carbon compounds for p gluconeogenesis

14 Role of PDC in the Fed State Glucose Pyr PDC Acetyl-CoA CAC Glycogen TAG FFA VLDL Glucose Pyr Glycogen PDC Acetyl-CoA CO 2 FFA CAC Adipose TAG CO22

15 Role of PDC during fasting Lac Glc Glucose Pyr PDC Acetyl-CoA KBs Pyr PDC Acetyl-CoA Lac FOX FFAs CAC Re-esterification Lipolysis Adipose

16 Role of PDC in diabetes Lac Glc Glucose Protein Pyr PDC Acetyl-CoA KBs Pyr PDC Acetyl-CoA Lac FOX FFAs CAC Re-esterification Lipolysis Adipose

17 Hypothesis Up regulation of PDK4 expression is required for conservation of three carbon substrates during fasting. Prediction: PDK4 KO mouse will have a lower than normal fasting blood glucose level.

18 Blood glucose levels in WT and PDK4 KO mice Metabolic State Blood glucose WT PDK4 -/- mg/dl Fd Fed 140 ± ± 10 Fasted overnight 64 ± 3 45 ± 1* *P<0.01

19 Why does knocking out PDK4 produce lower than normal fasting blood levels of glucose? Hypothesis: Low levels of blood three-carbon compounds limit glucose synthesis by the liver

20 Blood Levels of Three Carbon Compounds in WT and KO Mice Measurements PDK4 +/+ mice Blood from: PDK4 -/- mice Lactate (mmol/l) 1.93 ± ± 0.10* Pyruvate (mmol/l) 007± 0.07 ± ± 0.03 ± 001* 0.01 Alanine (mmol/l) 0.26 ± ± 0.01* *P<0.01

21 PDK4 KO mice are able to survive long term starvation After 48 hours of starvation: Blood glucose: 58 ± 3 mg/dl Blood acetoacetate: 2.2 ± 0.1 mm Blood β-hydroxybutyrate: 6.4 ± 1.8 mm Mice remain active and appear normal.

22 PDK4 expression is up regulated in mice rendered diabetic with a high fat diet Contributes to the hyperglycemia of mice fed a high fat diet

23 A Skeletal muscle B Diaphragm Chow HF Chow HF PDK4 PDK4 VDAC1α VDAC1α PDK4 (Fold) * PDK4 (Fold) * 0 0 PDK4 +/+ Chow fed PDK4 +/+ HF fed PDK4 +/+ Chow fed PDK4 +/+ HF fed Figure 2, Jeoung, et. al.

24 Knocking out PDK4 lowers blood glucose in mice rendered d diabetic with a high fat diet

25 Blood Analyte Levels in Wild Type and PDK4 Knockout Mice Fed High Fat Diet Measurements Blood from: PDK4 +/+ mice PDK4 -/- mice Glucose (mg/dl) 109 ± 7 87 ± 4 * Lactate (mmol/l) 27± 2.7 ± ± 2.1 ± * Pyruvate (mmol/l) 0.16 ± ± 0.01 * Alanine (mmol/l) 025± 0.25 ± ± 0.12 ± * FFA (mmol/l) 0.40 ± ± 0.06 * β-hydroxybutyrate (mmol/l) 098± 0.98 ± ± 2.29 ± * Acetoacetate (mmol/l) 0.18 ± ± 0.08 * Branched chain amino acids (mmol/l) 023± 0.23 ± ± 0.31 ± * Branched chain keto acids (mmol/l) ± ± * Triacylglycerols (mg/dl) 55 ± 4 88 ± 6 * Glycerol (mmol/l) 0.54 ± ± 0.07

26 Knocking out PDK4 reduces liver fat accumulation

27 PDK4 KO mice accumulate less fat in their livers Wild type liver PDK4 KO liver Sections stained with oil red O for fat

28 What about PDK2? PDK2 constitutively expressed in most tissues PDK2 expression increased in liver and kidney in response to fasting and diabetes We therefore generated PDK2 knockout mouse to determine the effect on glucose homeostasis.

29 Summary of effects of knocking out PDK2 Mice are viable. No effect on growth. No effect on fed or fasting blood glucose levels. No effect on glucose tolerance test.

30 PDK2/PDK4 double knockout mouse Up regulation of PDK2 may compensate for PDK4 in the PDK4 knockout mouse. Up regulation of PDK4 may compensate for PDK2 in the PDK2 knockout mouse. Therefore we produced d DKO mice.

31 Activity State of PDC in Skeletal Muscle of Fasted WT and PDK2/PDK4 DKO Mice Genotype %Active WT 2.8 ± 0.4 DKO 68.4 ± 0.5* N = 4 mice/group; *P<0.01

32 Overnight fasting blood analytes of WT and DKO mice Measurements Wild Type Blood from: DKO mice Glucose (mg/dl) 60 ± 2 36 ± 1 ** Lactate (mmol/l) 2.39 ± ± 0.22 * Pyruvate (mmol/l) ± ± * Alanine (mmol/l) 0.22 ± ± 0.02 * β-hydroxybutyrate (mmol/l) 0.92 ± ± 0.61 * Acetoacetate (mmol/l) 0.17 ± ± 0.13 ** Branched chain amino acids (mmol/l) 0.36 ± ± 0.03 *** Branched chain α-ketoacids (mmol/l) ± ± 0.003* Free fatty acids (mmol/l) 1.30 ± ± 0.37 Triacylglycerol (mg/dl) 103 ± ± 32 Glycerol (mmol/l) 0.31 ± ± 0.05 [β-hydroxybutyrate]/[ Acetoacetate] 55± 5.5 ± ± 2.3 ± ** *P < 0.05, **P<0.005, **P<0.0005

33 Body weight and blood glucose in WT and DKO mice fed a high h fat diet Genotype WT DKO Body weight g Blood glucose mg/di Yasmeen Rahimi and Byounghoon Hwang

34 Serum metabolites of wild-type and DKO mice fed a high fat diet Analyte Units Wildtype DKO Blood glucose mg/di Lactate mmol/l Hydroxybutyrate mmol/l Insulin ng/ml Triglyceride mg/di * * *

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36 Histology of livers from WT and DKO on a high saturated fat diet oil red O staining WT DKO Macrovesicular steatosis Microvesicular steatosis Yasmeen Rahimi

37 Effect of Knocking out PDK2 and PDK4 on Lipogenic Enzyme Expression WT DOKO PDK2 PDK4 ACC FAS Yasmeen Rahimi

38 Role of PDK2 & 4 during starvation Glc Glc Lac Pyr PDK2 & PDK4 Pyr Lac PDC PDC Acetyl-CoA KBs Acetyl-CoA FFAs CAC Re-esterification Lipolysis Adipose

39 Effects of knocking out PDK2 & 4 Glc Glc Lac Pyr PDC Acetyl-CoA PDK2 & PDK4 KBs Pyr PDC Acetyl-CoA Lac FFAs CAC Lipolysis Adipose

40 DKO mice can not survive long term starvation Mice suffer severe ketoacidosis and hypoglycemia if starved longer than overnight. This stands in contrast to PDK2 KO and PDK4 KO mice which readily tolerate 48 hours of starvation. Nevertheless, this demonstrates the critical importance of regulation of PDC activity by PDKs during starvation.

41 Potential problems with using PDK inhibitors to lower blood glucose Cause hypoglycemia Cause ketoacidosis Measurement Wild type DoKO Glucose (mg/dl) 60 ± 2 36 ± 1 Ketone bodies (mm) 11± ±

42 Effect of starvation on blood ph and bicarbonate in in WT and DKO mice ph 7.60 WT DOKO Fed Starved /L) HCO 3 (mmol Fed Starved Ketoacidosis Yasmeen Rahimi

43 Possible reason why ketone bodies are increased in PDK knockout mice Ketone bodies are made in the liver and used in peripheral tissues Ketone body formation from pyruvate in the liver Inhibition of ketone body oxidation in peripheral tissues by pyruvate oxidation

44 Why accumulation of ketone bodies causes acidosis Palmitic acid + 23 O 2 16 CO H 2 O Palmitic acid + 5 O 2 4 β-hydroxybutyrate + 4 H + 4 β-hydroxybutyrate + 4 H O 2 16 CO H 2 O Excretion of β-hydroxybutyrate in urine leaves H + in blood

45 Ketone bodies are elevated by knocking out PDKs Is this as bad as it sounds? Ketone bodies are water-soluble fat calories (Hans Krebs) Ketone bodies increased by Atkins Diet without adverse effects Only partial inhibition of PDK activity may prove beneficial

46 Ketone body formation from glucose also cause acidosis? Glucose + 6 O 2 6 CO2 + 6 H2O Glucose 2 lactate +2H + Glucose O 2 β-hydroxybutyrate + H + +2CO2+2H2O + 2

47 Potential problems with using PDK inhibitors to treat diabetes Cause insulin resistance by inhibiting fatty acid oxidation in skeletal muscle?

48 Mechanism responsible for insulin resistance MCD FFA (palmitate) FAT FFA (palmitate) Insulin signaling Acetyl-CoA Malonyl-CoA Palmityl-CoA DAG, ceramide ACC-1 CPT-1 mitochondrion TAG Citrate β-oxidation PDK4 Acetyl-CoA PDH Pyruvate Citrate TCA cycle Glucose Pyruvate

49 Lipid accumulates in the heart of DOKO fed a high saturated fat diet WT DOKO Fatty Heart Yasmeen Rahimi

50 Why this may not be a problem Insulin sensitivity is slightly increased rather than decreased in the PDK KO mice

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53 Are the PDKs viable targets t for the treatment of Type 2 Diabetes? Compounds that t inhibit PDKs should: lower blood glucose improve glucose tolerance increase insulin sensitivity reduce hepatic steatosis decrease body fat

54 Are the PDKs viable targets for the treatment of Type 2 Diabetes? However, over inhibition of the PDKs: will induce hypoglycemia will induce ketoacidosis may induce fat accumulation in some tissues may induce insulin resistance

55 Good PDK inhibitor is needed! Many drug companies have tried and given up. AstraZenica compounds: AZD7545; Binds at the lipoyl binding site; inhibits PDK2 but activates PDK4. R-Lipoic acid many effects; difficult to sort out whether any of its effects are due to inhibition of the PDKs. Possible lead compound. Novartis compounds: Anilides of trifluoro-2-hydroxy-2- methylpropionic acid; Leelamine effective in intact cells but weak. Possible lead compound.

56 Finding a good PDK inhibitor Might be facilitated by a better understanding of the structure and function of the PDKs X-ray crystal structures have been produced of all four PDKs Information is accumulating relevant to the mechanisms responsible for regulation of the activities of the PDKs

57 Pyruvate Dehydrogenase Kinases Four isoforms (PDK1,2,3,4) that are expressed in a tissue specific manner. 70% identical. 30% identical to the branched chain keto acid dehydrogenase kinase. Structurally related to the bacterial histidine protein kinases. Completely different from the cytoplasmic protein kinases.

58 PDC OP ADP Inactive H 2 O DCA (-) (-) PDH (+) PDH P ase 1 Ca 2+ (-) (+) Kinases PDH (-) ,2,3,4 P ase 2 (-) Pi ATP CoASH Pyruvate NAD + PDC OH Active CO 2 Acetyl-CoA NADH

59 Some things we have learned about inhibition of PDC by the PDKs DCA and 2-CP mimic the inhibitory effects of pyruvate by binding in the pyruvate site. Compounds that bind in this site are all small. Pyruvate, DCA, and 2-CP increase the affinity for ADP and decrease the affinity for ATP. PDK3 is NOT sensitive to inhibition by pyruvate and its analogues.

60 PDK2 Structure ATP/ADP binding site C-terminal domains DCA binding site E2 Lipoyl group binding domain N-terminal domain Steussy et al. JBC 276: , 2001

61 Chuang Model for PDK Regulation DCA binding site Disordered C- terminal tails Ordered C-terminal cross tails Wynn et al. J. Biol. Chem. 283: , 2008

62 Summary Knocking out PDK4 results in lower fasting blood glucose levels and modestly improved glucose tolerance and insulin sensitivity Knocking out PDK4 results in less fat accumulation in the liver of high fat fed mice. Knocking out both PDK2 and PDK4 induces greater lowering of fasting blood glucose levels l and markedly improved glucose tolerance PDKs may be viable targets for the treatment of diabetes

63 Acknowledgements Nam Ho Jeoung Indianapolis and Daegu Byounghoon Hwang - Indianapolis Yasmeen Rahimi - Indianapolis Pengfei Wu - Indianapolis Professor In-Kyu Lee - Daegu

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65 Role of the PDC in Glucose Homeostasis Lactate Oxaloacetate Glucose Pyruvate PDC Acetyl-CoA Alanine Fat Glucose Citric acid cycle Ketone bodies PDC is active when we are well fed PDC is inactive when we are fasting or starved

66 Model for Regulation of the PDKs Active site fixed open for ATP Lipoyl groups of swinging arm of E2 Wynn et al. J. Biol. Chem. 283: , 2008

67 Some things we have learned about inhibition of PDC by the PDKs The PDKs show preferential binding to E2: PDK3>PDK1~PDK2>PDK4 PDK1 PDK2 PDK4 PDK2 and PDK3 have low basal activities but they are greatly activated t by binding to E2. PDK1 and PDK4 are NOT activated by binding to E2. These PDKs have HIGH basal activity.

68 Modern view as to why aerobic glycolysis gy y may be important in cancer Mitochondria are primarily responsible for ROS production. Hypoxia promotes ROS production by mitochondria. ROS induces apoptosis and limits growth. Inhibition of mitochondrial redox reactions decreases ROS production, decreases apoptosis, and allows growth of cancer cell.

69 How aerobic glycolysis is increased in cancer cells. Hypoxia induces HIF-1 which induces expression of glycolytic l enzymes. Tumor suppressor p53, which normally maintains low concentration of F2,6P 2 (activator of PFK1), is down regulated in cancer cells. Most recent finding: HIF-1 induces expression of PDK1 which inhibits PDC, which inhibits pyruvate oxidation, which inhibits ROS production.

70 Cancer Summary Increased capacity of cancer cells for aerobic glycolysis l is due in part to up regulation of PDK1 and PDK3. This protects cancer cells by reducing ROS generation Down regulation of PDK1 promotes complete oxidation of glucose by cancer cells and decreases growth PDKs may be viable targets for the treatment of cancer

71 Anilides of Trifluoro-2-hydroxy-2-2 methylpropionic yp p acid PDK inhibitors Activate PDC Not effective in lowering blood glucose levels References: (Novartis) Aicher et al. J. Med. Chem. (2000) Bebernitz et al. J. Med. Chem. (2000)