Open Access

Metabolomic profiling reveals a role for CPT1c in neuronal oxidative metabolism

BMC Biochemistry201213:23

DOI: 10.1186/1471-2091-13-23

Received: 20 August 2012

Accepted: 18 October 2012

Published: 25 October 2012

Abstract

Background

Carnitine Palmitoyltransferase-1c (CPT1c) is a neuron specific homologue of the carnitine acyltransferase family of enzymes. CPT1 isoenzymes transfer long chain acyl groups to carnitine. This constitutes a rate setting step for mitochondrial fatty acid beta-oxidation by facilitating the initial step in acyl transfer to the mitochondrial matrix. In general, neurons do not heavily utilize fatty acids for bioenergetic needs and definitive enzymatic activity has been unable to be demonstrated for CPT1c. Although there are studies suggesting an enzymatic role of CPT1c, its role in neurochemistry remains elusive.

Results

In order to better understand how CPT1c functions in neural metabolism, we performed unbiased metabolomic profiling on wild-type (WT) and CPT1c knockout (KO) mouse brains. Consistent with the notion that CPT1c is not involved in fatty acid beta-oxidation, there were no changes in metabolites associated with fatty acid oxidation. Endocannabinoids were suppressed in the CPT1c KO, which may explain the suppression of food intake seen in CPT1c KO mice. Although products of beta-oxidation were unchanged, small changes in carnitine and carnitine metabolites were observed. Finally, we observed changes in redox homeostasis including a greater than 2-fold increase in oxidized glutathione. This indicates that CPT1c may play a role in neural oxidative metabolism.

Conclusions

Steady-state metabolomic analysis of CPT1c WT and KO mouse brains identified a small number of metabolites that differed between CPT1c WT and KO mice. The subtle changes in a broad range of metabolites in vivo indicate that CPT1c does not play a significant or required role in fatty acid oxidation; however, it could play an alternative role in neuronal oxidative metabolism.

Background

Although the mammalian brain is lipid rich and mutations in lipid metabolizing enzymes result in debilitating neurological disease, neurons are generally not thought to rely on mitochondrial fatty acid beta-oxidation for bioenergetic requirements. Neurons instead mainly utilize the oxidation of glucose for most of their bioenergetic needs, although, during prolonged fasting, ketone bodies (i.e. acetoacetate and beta hydroxybutyrate) can also be used [1]. Most neurons have a low amount of the rate-setting enzymes in mitochondrial long chain fatty acid catabolism, namely, the malonyl-CoA sensitive Carnitine Palmitoyltransferase 1 (CPT1a and CPT1b) enzymes which limit most neurons potential for mitochondrial fatty acid beta-oxidation [2].

Carnitine acyltransferases are enzymes that catalyze the exchange of acyl groups between carnitine and Coenzyme A (CoA) to facilitate the transport acyl chains between the cytoplasm to the mitochondrial matrix [3]. CPT1 isoenzymes (EC 2.3.1.21) preferentially are positioned on the outer mitochondrial membrane and transfer long chain acyl groups from CoA to carnitine. CPT1a and CPT1b are malonyl-CoA sensitive and therefore inhibited when malonyl-CoA levels are high (e.g. during high glucose flux). The malonyl-CoA insensitive CPT2, on the other hand, is located in the mitochondrial matrix and reversibly transfers the acyl chain back to CoA to facilitate beta-oxidation. Although neurons have a relative dearth of CPT1a and CPT1b [2], they express a CPT1 homologue, CPT1c [4].

CPT1c has a high primary amino acid sequence similarity and identity to the canonical CPT enzymes. Therefore, it was surprising that definitive acyltransferase activity or enhanced oxidation of fatty acids could not be shown for CPT1c [46]. CPT1c KO mice exhibit both behavioral and metabolic deficits [69]. Over-expression of CPT1c in the brain of developing transgenic mice results in microencephaly [10]. Therefore, it is clear that CPT1c plays an important role in brain function. Although there were several metabolites identified that have been altered after over-expression [10, 11] or knockout of CPT1c [7], the reaction that CPT1c catalyzes has remained elusive.

Here we used an unbiased metabolomic approach to broadly understand the consequence of CPT1c deletion to gain insight into the biochemical and physiological roles of CPT1c function. Similar to previous work in heterologous systems, we did not see changes consistent with a role for CPT1c in long chain fatty acid beta oxidation. However, there were changes in several fatty acid derived metabolites including endocannabinoids, which may explain the suppressed food intake in these models. Also, some of the most abundant changes were in redox biochemistry consistent with several models of CPT1c function recently proposed.

Methods

Animals

Mice with a targeted knockout of exons 1 and 2 of the cpt1c gene were propagated and genotyped as previously described [5, 6]. Mice were fed a standard lab chow (Harlan 2018) after weaning. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and under the approval of the Johns Hopkins Medical School Animal Care and Use Committee.

Western blot analysis

A polyclonal rabbit antibody against CPT1c was used as a primary antibody for CPT1c detection in WT and CPT1c KO mice [5, 6]. Anti-rabbit horseradish peroxidase (HRP) was used as a secondary antibody, and the blots for CPT1c were developed using ECL reagent. Mouse monoclonal anti-HSC70 (Santa Cruz biotech) and mouse monoclonal anti beta-actin (Sigma) was used as primary antibodies for loading control. Cy3 conjugated fluorescent secondary antibody was used for both HSC70 and beta-actin antibodies.

Metabolomic measurements and profiling

Unbiased metabolomics analysis of whole brain samples from WT and CPT1c KO mice (n=8/group) that were fasted overnight was performed using liquid chromatography/tandem mass spectrometry (HPLC/MS/MS2) and gas chromatography/mass spectrometry (GC/MS) platforms. The platform was able to screen and identify several metabolites in multiple classes, such as amino acids, lipids, and nucleotides. A complete list of the metabolites identified in this study is given in Tables 1, 2, 3 and 4. General platform methods about metabolomic measurements and profiling are described in the metabolomic study done by Eckel-Mahan et al. [12]
Table 1

Biochemicals involved in lipid metabolic pathways

PATHWAY

SUB PATHWAY

BIOCHEMICAL NAME

KEGG

CPT1c KO CPT1c WT

Welch's Two-Samplet-Test

CAS

PUBCHEM

Lipid

Essential fatty acid

linoleate (18:2n6)

C01595

0.93

0.4643

60-33-3;

5280450

linolenate [alpha or gamma; (18:3n3 or 6)]

C06427

1.04

0.4808

  

dihomo-linolenate (20:3n3 or n6)

C03242

0.81

0.0608

 

5312529

eicosapentaenoate (EPA; 20:5n3)

C06428

0.72

0.0236

10-2005-9;10417-94-4;

446284

docosapentaenoate (n3 DPA; 22:5n3)

C16513

0.80

0.0662

2234-74-4;

 

docosapentaenoate (n6 DPA; 22:5n6)

C06429

0.77

0.3030

25182-74-5;

6441454

docosahexaenoate (DHA; 22:6n3)

C06429

0.89

0.2879

6217-54-5;

445580

Medium chain fatty acid

caproate (6:0)

C01585

0.98

0.5408

142-62-1;

8892

caprylate (8:0)

C06423

0.99

0.9309

124-07-2;

379

pelargonate (9:0)

C01601

0.89

0.1531

112-05-0;

5461016

laurate (12:0)

C02679

1.01

0.9051

143-07-7;

3893

Long chain fatty acid

myristate (14:0)

C06424

0.99

0.8942

544-63-8;

11005

myristoleate (14:1n5)

C08322

1.26

0.1786

544-64-9;

5281119

palmitate (16:0)

C00249

0.85

0.0781

57-10-3;

985

palmitoleate (16:1n7)

C08362

0.93

0.3794

373-49-9;

445638

margarate (17:0)

 

0.86

0.2288

506-12-7;

10465

10-heptadecenoate (17:1n7)

 

0.81

0.1051

29743-97-3;

5312435

stearate (18:0)

C01530

0.94

0.4536

57-11-4;

5281

oleate (18:1n9)

C00712

0.88

0.2434

112-80-1;

445639

10-nonadecenoate (19:1n9)

 

0.72

0.0470

73033-09-7;

5312513

eicosenoate (20:1n9 or 11)

 

0.78

0.1453

  

dihomo-linoleate (20:2n6)

C16525

0.75

0.0804

2091-39-6;

6439848

arachidonate (20:4n6)

C00219

0.94

0.4832

506-32-1;

444899

docosadienoate (22:2n6)

C16533

0.84

0.3185

7370-49-2;

5282807

adrenate (22:4n6)

C16527

0.81

0.1467

2091-25-0;

5282844

Fatty acid, ester

n-Butyl Oleate

 

0.96

0.7046

142-77-8;

5354342

Fatty acid, dicarboxylate

2-hydroxyglutarate

C02630

1.00

0.8616

40951-21-1;

43

Fatty acid, amide

oleamide

 

1.22

0.7962

301-02-0;

5283387

stearamide

C13846

1.19

0.6546

124-26-5;

31292

Eicosanoid

prostaglandin D2

C00696

1.20

0.1092

41598-07-6;

448457

prostaglandin E2

C00584

0.93

0.3928

363-24-6;

5280360

5-HETE

 

0.99

0.8472

73307-52-5;

9862886

15-HETE

C04742

0.83

0.9669

54845-95-3;

5280724

Endocannabinoid

palmitoyl ethanolamide

 

0.64

0.0331

 

4671

Fatty acid & BCAA metabolism

propionylcarnitine

C03017

1.08

0.4494

17298-37-2;

107738

Carnitine metabolism

carnitine

C00487

0.88

0.0084

461-05-2;

288

3-dehydrocarnitine*

C02636

1.22

0.0103

10457-99-5;

6991982

acetylcarnitine

C02571

0.89

0.2172

5080-50-2;

7045767

oleoylcarnitine

 

0.83

0.3694

  

Fatty alcohol, long chain

1-octadecanol

D01924

1.01

0.8513

112-92-5;

8221

Glycerolipid metabolism

choline phosphate

C00588

0.97

0.4914

72556-74-2;

1014

ethanolamine

C00189

1.10

0.4703

141-43-5;

 

phosphoethanolamine

C00346

1.06

0.5812

1071-23-4;

52,323,241,015

glycerol

C00116

0.97

0.6203

56-81-5;

753

glycerol 3-phosphate (G3P)

C00093

0.98

0.6926

29849-82-9;

754

glycerophosphorylcholine (GPC)

C00670

0.96

0.9071

28319-77-9;

657272

cytidine 5'-diphosphocholine

C00307

1.25

0.0583

33818-15-4;

13805

Inositol metabolism

myo-inositol

C00137

0.94

0.0882

87-89-8;

 

chiro-inositol

 

0.77

0.1568

643-12-9;

 

inositol 1-phosphate (I1P)

 

1.01

0.8432

106032-59-1;

 

scyllo-inositol

C06153

0.90

0.1635

488-59-5;

 

Ketone bodies

3-hydroxybutyrate (BHBA)

C01089

1.23

0.2197

625-72-9;

441

Lysolipid

1-palmitoylglycerophosphoethanolamine

 

1.12

0.8591

 

9547069

2-palmitoylglycerophosphoethanolamine*

 

0.83

0.1926

  

1-stearoylglycerophosphoethanolamine

 

1.20

0.7882

69747-55-3;

9547068

1-oleoylglycerophosphoethanolamine

 

1.14

0.8654

 

9547071

2-oleoylglycerophosphoethanolamine*

 

1.07

0.9602

  

1-arachidonoylglycerophosphoethanolamine*

 

1.06

0.8488

  

2-arachidonoylglycerophosphoethanolamine*

 

0.45

0.2213

  

2-docosahexaenoylglycerophosphoethanolamine*

 

0.48

0.3141

  

1-palmitoylglycerophosphocholine

 

0.47

0.1450

17364-16-8;

86554

2-palmitoylglycerophosphocholine*

 

0.59

0.2106

  

1-stearoylglycerophosphocholine

 

0.51

0.1452

19420-57-6;

497299

2-stearoylglycerophosphocholine*

 

1.00

  

10208382

1-oleoylglycerophosphocholine

 

0.56

0.1923

19420-56-5;

16081932

2-oleoylglycerophosphocholine*

 

0.65

0.3441

  

1-arachidonoylglycerophosphocholine*

C05208

1.00

   

2-arachidonoylglycerophosphocholine*

 

0.89

0.4485

  

1-docosahexaenoylglycerophosphocholine*

 

1.00

   

2-docosahexaenoylglycerophosphocholine*

 

0.86

0.4614

  

1-palmitoylglycerophosphoinositol*

 

0.85

0.2160

  

1-stearoylglycerophosphoinositol

 

0.77

0.1315

  

1-arachidonoylglycerophosphoinositol*

 

0.87

0.3521

  

1-oleoylglycerophosphoserine

 

0.92

0.6515

 

9547099

2-oleoylglycerophosphoserine*

 

0.80

0.1921

  

1-palmitoylplasmenylethanolamine*

 

1.23

0.5225

  

Monoacylglycerol

1-palmitoylglycerol (1-monopalmitin)

 

0.83

0.1685

542-44-9;

14900

1-stearoylglycerol (1-monostearin)

D01947

0.92

0.3625

123-94-4;

24699

2-stearoylglycerol (2-monostearin)

 

0.75

0.1774

621-61-4;

79075

1-oleoylglycerol (1-monoolein)

 

0.80

0.1139

111-03-5;

5283468

2-oleoylglycerol (2-monoolein)

 

0.59

0.0769

3443-84-3;

5319879

Sphingolipid

sphingosine

C00319

0.71

0.3009

123-78-4;

5353955

palmitoyl sphingomyelin

 

0.84

0.1297

 

9939941

stearoyl sphingomyelin

C00550

1.07

0.2147

85187-10-6;85187-10-6;

6453725

Mevalonate metabolism

3-hydroxy-3-methylglutarate

C03761

1.07

0.4426

503-49-1;

5459993

Sterol/Steroid

cholesterol

C00187

1.00

0.9987

57-88-5;

6432564

7-alpha-hydroxycholesterol

C03594

1.24

0.2998

566-27-8;

107722

7-beta-hydroxycholesterol

C03594

1.11

0.2969

566-27-8;

473141

24(S)-hydroxycholesterol

C13550

0.94

0.5728

2140-46-7;

 

corticosterone

C02140

0.59

0.2402

50-22-6;

5753

Table 2

Biochemicals in the amino acid and peptide pathways

PATHWAY

SUB PATHWAY

BIOCHEMICAL NAME

KEGG

CPT1c KO CPT1c WT

Welch's Two-Samplet-Test

CAS

PUBCHEM

Amino acid

Glycine, serine and threonine metabolism

glycine

C00037

0.91

0.1984

56-40-6;

5,257,127,750

serine

C00065

0.98

0.6400

56-45-1;

59,516,857,581

N-acetylserine

 

1.16

0.2513

97-14-3;

65249

homoserine

C00263,C02926

1.04

0.5460

672-15-1;

126,476,971,022

3-phosphoserine

C01005

1.06

0.4516

407-41-0;

 

threonine

C00188

0.98

0.8340

72-19-5;

69,710,196,288

allo-threonine

C05519

0.98

0.7264

28954-12-3;

992,896,995,276

betaine

C00719

0.62

0.0393

107-43-7;

247

Alanine and aspartate metabolism

alanine

C00041

0.99

0.8540

56-41-7;

59,507,311,724

beta-alanine

C00099

0.95

0.7707

56-41-7;107-95-9;

2,394,755,801

N-acetylalanine

C02847

0.96

0.7172

97-69-8;

88064

aspartate

C00049

1.02

0.5759

56-84-8;

5960

N-acetylaspartate (NAA)

C01042

0.98

0.7849

997-55-7;997-55-7;

65065

Glutamate metabolism

glutamate

C00025

1.10

0.1218

56-86-0;

611

glutamine

C00064

0.96

0.3866

56-85-9;

69,920,865,961

gamma-aminobutyrate (GABA)

C00334

1.07

0.4581

56-12-2;

6,992,099,119

N-acetylglutamate

C00624

1.21

0.1108

5817-08-3;

1549099

N-acetyl-aspartyl-glutamate (NAAG)

C12270

1.04

0.6033

3106-85-2;

5255

N-acetylglutamine

C02716

0.79

0.1871

2490-97-3;

182230

Histidine metabolism

histidine

C00135

1.11

0.1815

5934-29-2;

7,733,651,426

Lysine metabolism

lysine

C00047

0.81

0.0655

56-87-1;

5962

2-aminoadipate

C00956

0.99

0.9856

542-32-5;1118-90-7;

469

pipecolate

C00408

0.91

0.4383

4043-87-2;

849

glutaroyl carnitine

 

0.77

0.0244

102636-82-8;

 

Phenylalanine & tyrosine metabolism

phenylalanine

C00079

0.93

0.0731

63-91-2;

69,256,656,140

tyrosine

C00082

1.10

0.1569

60-18-4;

60,576,942,100

3-(4-hydroxyphenyl)lactate

C03672

1.28

0.1580

6482-98-0;

9378

Tryptophan metabolism

tryptophan

C00078

1.10

0.1009

73-22-3;

69,235,166,305

C-glycosyltryptophan*

 

1.00

0.9578

  

5-hydroxyindoleacetate

C05635

0.99

0.9982

54-16-0;

1826

Valine, leucine and isoleucine metabolism

isoleucine

C00407

0.99

0.7705

73-32-5;

791

leucine

C00123

0.92

0.1061

61-90-5;

70,457,986,106

valine

C00183

1.00

0.9896

72-18-4;

69,710,186,287

alpha-hydroxyisovalerate

 

0.92

0.9081

600-37-3;

99823

2-methylbutyroylcarnitine

 

0.98

0.7985

31023-25-3;

6426901

isovalerylcarnitine

 

0.90

0.1479

 

6426851

hydroxyisovaleroyl carnitine

 

0.90

0.0807

99159-87-2;

 

Cysteine, methionine, SAM, taurine metabolism

cysteine

C00097

1.13

0.0835

52-90-4;56-89-3;

58,626,419,722

cystine

C00491

0.86

0.5529

56-89-3;

595

taurine

C00245

1.03

0.7783

107-35-7;

11,234,068,592

S-adenosylhomocysteine (SAH)

C00021

0.96

0.4778

979-92-0;

 

methionine

C00073

0.95

0.1654

63-68-3;

69,920,876,137

N-acetylmethionine

C02712

0.88

0.1362

65-82-7;

448580

2-hydroxybutyrate (AHB)

C05984

1.23

0.5077

3347-90-8;

440864

Urea cycle; arginine-, proline-, metabolism

arginine

C00062

0.95

0.0964

1119-34-2;

5,246,487,232

ornithine

C00077

0.90

0.2453

3184-13-2;

6262

urea

C00086

0.71

0.2913

57-13-6;

117,616,150,869

proline

C00148

0.97

0.6099

147-85-3;

1,457,426,971,047

N-acetylornithine

C00437

1.26

0.3497

6205-08-9;

6,992,102,439,232

trans-4-hydroxyproline

C01157

1.03

0.6431

51-35-4;

58,106,971,053

argininosuccinate

C03406

0.86

0.3803

156637-58-0;

828

Creatine metabolism

creatine

C00300

1.03

0.2564

57-00-1;

586

creatinine

C00791

1.20

0.1694

60-27-5;

588

Butanoate metabolism

2-aminobutyrate

C02261

1.03

0.8503

1492-24-6;

4,396,916,971,251

Polyamine metabolism

5-methylthioadenosine (MTA)

C00170

1.08

0.2023

2457-80-9;

439176

putrescine

C00134

0.83

0.4688

110-60-1;

 

spermidine

C00315

1.04

0.6645

124-20-9;

1102

spermine

C00750

0.99

0.4470

71-44-3;

1103

Guanidino and acetamido metabolism

4-guanidinobutanoate

C01035

0.98

0.7911

463-003;463-00-3;

500

Glutathione metabolism

glutathione, reduced (GSH)

C00051

1.53

0.1024

70-18-8;

124886

5-oxoproline

C01879

0.86

0.0291

98-79-3;

7405

glutathione, oxidized (GSSG)

C00127

2.15

0.0307

103239-24-3;

6,535,911,215,652

cysteine-glutathione disulfide

 

1.33

0.0802

13081-14-6;

4247235

Peptide

Dipeptide derivative

carnosine

C00386

0.98

0.8057

305-84-0;

4,392,246,992,100

homocarnosine

C00884

1.00

0.9807

3650-73-5;

10243361

gamma-glutamyl

gamma-glutamylleucine

 

0.91

0.1529

2566-39-4;

151023

gamma-glutamylglutamate

 

1.24

0.1880

1116-22-9;

92865

gamma-glutamylglutamine

 

0.93

0.4450

10148-81-9;

150914

gamma-glutamylphenylalanine

 

0.93

0.5544

7432-24-8;

111299

Table 3

Biochemicals from the carbohydrate and energy pathways

PATHWAY

SUB PATHWAY

BIOCHEMICAL NAME

KEGG

CPT1c KO CPT1c WT

Welch's Two-Samplet-Test

CAS

PUBCHEM

Carbohydrate

Aminosugars metabolism

N-acetylglucosamine

C00140

1.03

0.7477

7512-17-6;

24139

erythronate*

 

0.98

0.7434

13752-84-6;

2781043

N-acetylneuraminate

C00270

1.03

0.4494

131-48-6;

 

Fructose, mannose, galactose, starch, and sucrose metabolism

fructose

C00095

0.98

0.8393

57-48-7;

5984

mannose

C00159

0.94

0.6417

3458-28-4;

161658

mannose-6-phosphate

C00275

0.97

0.7187

70442-25-0;104872-94-8;

 

sorbitol

C00794

0.92

0.5926

6706-59-8;

107428

Glycolysis, gluconeogenesis, pyruvate metabolism

1,5-anhydroglucitol (1,5-AG)

C07326

0.95

0.7426

154-58-5;

 

glycerate

C00258

0.96

0.4928

600-19-1;

752

glucose-6-phosphate (G6P)

C00668

0.96

0.5074

103192-55-8;

 

glucose

C00293

0.86

0.1984

50-99-7;

79025

fructose-6-phosphate

C05345

0.83

0.1261

103213-47-4;

 

Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate

 

0.98

0.8050

  

3-phosphoglycerate

C00597

0.80

0.1220

80731-10-8;

 

dihydroxyacetone phosphate (DHAP)

C00111

1.02

0.6910

102783-56-2;

4643300

1,3-dihydroxyacetone

C00184

1.12

0.4601

62147-49-3;

670

pyruvate

C00022

0.83

0.0193

127-17-3;

107735

lactate

C00186

1.06

0.3677

79-33-4;

612

Nucleotide sugars, pentose metabolism

arabitol

C00474

1.30

0.0435

488-82-4;

94154

ribitol

C00474

0.86

0.1732

488-81-3;

 

sedoheptulose-7-phosphate

C05382

0.91

0.4130

2646-35-7;

616

ribose 5-phosphate

C00117

1.39

0.0353

18265-46-8;108321-05-7;

447634

Isobar: ribulose 5-phosphate, xylulose 5-phosphate

 

1.06

0.5400

  

arabinose

C00181

1.08

0.5432

28697-53-2;

66308

Energy

Krebs cycle

citrate

C00158

1.02

0.5785

77-92-9;

311

alpha-ketoglutarate

C00026

0.79

0.2702

305-72-6;328-50-7;22202-68-2;

51

succinate

C00042

0.88

0.5010

110-15-6;

1110

fumarate

C00122

0.94

0.5055

100-17-8;

 

malate

C00149

1.11

0.2256

6915-15-7;

525

Oxidative phosphorylation

phosphate

C00009

0.98

0.3284

7664-38-2;

1061

pyrophosphate (PPi)

C00013

0.84

0.4801

1466-09-3;

644102

Table 4

Biochemicals in nucleotide, cofactors and vitamins, and xenobiotic Pathways

PATHWAY

SUB PATHWAY

BIOCHEMICAL NAME

KEGG

CPT1c KO CPT1c WT

Welch's Two-Samplet-Test

CAS

PUBCHEM

Nucleotide

Purine metabolism, (hypo)xanthine/inosine containing

xanthine

C00385

1.02

0.7727

69-89-6;

1188

hypoxanthine

C00262

0.98

0.4343

68-94-0;

790

inosine

 

1.00

0.8754

58-63-9;

 

Purine metabolism, adenine containing

adenine

C00147

1.11

0.0801

73-24-5;

190

adenosine

C00212

0.86

0.1407

58-61-7;

60961

N1-methyladenosine

C02494

0.94

0.3601

15763-06-1;

5460178

adenosine 2'-monophosphate (2'-AMP)

C00946

1.00

 

130-49-4;

 

adenosine 5'-monophosphate (AMP)

C00020

0.89

0.2000

149022-20-8;

15938965

Purine metabolism, guanine containing

guanosine

C00387

1.01

0.9130

118-00-3;

6802

Purine metabolism, urate metabolism

urate

C00366

1.06

0.4983

69-93-2;120K5305;

 

allantoin

C02350

0.76

0.1685

97-59-6;

204

Pyrimidine metabolism, cytidine containing

cytidine

C00475

0.94

0.1562

65-46-3;

6175

cytidine 5'-monophosphate (5'-CMP)

C00055

1.01

0.8988

63-37-6;

7058165

Pyrimidine metabolism, orotate containing

orotate

C00295

0.86

0.2325

50887-69-9;

967

Pyrimidine metabolism, uracil containing

uracil

C00106

0.97

0.5212

66-22-8;

1174

uridine

C00299

0.91

0.0141

58-96-8;

6029

pseudouridine

C02067

0.99

0.7648

1445-07-4;

 

Purine and pyrimidine metabolism

methylphosphate

 

0.85

0.1460

7023-27-0;

13130

Cofactors and vitamins

Ascorbate and aldarate metabolism

ascorbate (Vitamin C)

C00072

0.87

0.1924

134-03-2;

 

dehydroascorbate

C05422

1.70

0.2338

490-83-5;

835

threonate

C01620

0.96

0.5529

70753-61-6;

151152

Hemoglobin and porphyrin

heme*

C00032

0.69

0.3695

14875-96-8;

 

Nicotinate and nicotinamide metabolism

nicotinamide

C00153

1.00

0.9275

98-92-0;

936

nicotinamide adenine dinucleotide (NAD+)

C00003

0.87

0.0469

53-84-9;

1,089,765,158,925,280,000

Pantothenate and CoA metabolism

pantothenate

C00864

0.94

0.7951

137-08-6;

6613

phosphopantetheine

C01134

0.85

0.0841

NA;

115254

Pyridoxal metabolism

pyridoxal

C00250

1.05

0.5803

65-22-5;

1050

Riboflavin metabolism

flavin adenine dinucleotide (FAD)

C00016

0.93

0.1085

146-14-5;84366-81-4;

643975

riboflavin (Vitamin B2)

C00255

0.93

0.2187

83-88-5;

493570

flavin mononucleotide (FMN)

C00061

0.96

0.7167

130-40-5;

710

Tocopherol metabolism

alpha-tocopherol

C02477

1.04

0.6234

59-02-9;10191-41-0;

14985

Xenobiotics

Chemical

glycolate (hydroxyacetate)

C00160

1.06

0.7194

79-14-1;

3,698,251,757

glycerol 2-phosphate

C02979,D01488

1.02

0.9683

819-83-0;

2526

2-phenoxyethanol

 

0.94

0.9231

122-99-6;

 

2-pyrrolidinone

 

0.84

0.6590

616-45-5;

12025

Food component/Plant

ergothioneine

C05570

0.88

0.0968

58511-63-0;

3032311

Sugar, sugar substitute, starch

erythritol

C00503

0.89

0.0966

149-32-6;

 

Statistical analysis

Pair-wise comparisons between CPT1c WT and KO were performed using Welch’s two-sample t-tests. From the p-values, any value below the significance level of 0.05 was interpreted as statistically significant.

Results

Carnitine Palmitoyltransferase-1c KO mice

Although CPT1c is widely expressed in transformed cells and tumors [13], we have only been able to reliably detect CPT1c in neurons in vivo. To understand the endogenous function of CPT1c, we performed metabolomic profiling on brains of CPT1c KO mice and their littermate controls. Therefore, we collected and snap froze the brains of CPT1c KO and WT littermate sex matched adult mice after an overnight fast. Western blot analysis of WT and CPT1c KO mice showed that KO mice were indeed completely deficient of CPT1c (Figure 1A). These samples were then homogenized and the small organic metabolites were extracted and analyzed by a mixture of GC-MS and LC-MS/MS by a commercial supplier of metabolomic analyses (Figure 1B). Below, we detail the changes in steady-state biochemicals between WT and KO brains that were identified through an unbiased metabolomic screen.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_Article_396_Fig1_HTML.jpg
Figure 1

CPT1c KO mice and metabolomic profiling. (A) CPT1c protein from homogenized brains of WT and CPT1c KO mice were analyzed by western blot using the anti-CPT1c antibody. Hsc70 and Actin were used for loading controls. (B) A schematic pathway of metabolomic profiling for KO and WT brains. A commercial supplier of metabolic analysis homogenized 8 brain samples from independent mice to extract organic metabolites for performing unbiased metabolomic analysis using a mixture of GC-MS and LC-MS/MS.

Fatty acid oxidative metabolites show no difference in overall trend in CPT1c KO mice

Given the high primary amino acid homology of CPT1c to other CPTs, it would follow that CPT1c may be involved in fatty acid beta oxidation or at least in long chain acyl-CoA metabolism. If CPT1c was involved in fatty acid oxidation, we would expect that the deletion of CPT1c would decrease the level of acyl-carnitines and potentially increase the levels of other long chain acyl-CoA dependent biosyntheses. A broad range of lipid species were identified in the metabolomic screen (Table 1). No changes were seen in oleoyl-carnitine, beta-hydroxybutyrate, or acetyl-carnitine, as we would have expected (Figure 2A). However, the metabolomic analysis did show that free carnitine, 3-dehydrocarnitine, glutaroylcarnitine, and betaine were significantly changed (Figure 2A).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_Article_396_Fig2_HTML.jpg
Figure 2

Loss of CPT1c results in decreased free carnitine and no change in fatty acid oxidative metabolites in the brain. (A) Biochemicals involved in carnitine, amino acid, and fatty acid metabolism from WT and CPT1c KO brains were compared through metabolomic analyses, revealing a statistically significant change in levels of free carnitine (p=0.084), 3-dehydrocarnitine (p=0.0103), glutaroylcarnitine (p=0.0244) and betaine (p=0.0383). (B) Schematic of biochemical pathways altered in CPT1c KO mice. Based on this schematic pathway, glutaroyl carnitine and betaine may affect the level of free carnitine, since these biochemicals play a role in carnitine biosynthesis.

Among the metabolites that showed a statistically significant difference, only 3-dehydrocarnitine increased in CPT1c KO mice while glutaroyl carnitine, betaine and free carnitine decreased. Glutaroyl carnitine and betaine are biochemicals that are involved in carnitine biosynthesis (Figure 2B; Table 2). Glutaroyl carnitine is involved in lysine metabolism, which is one of the amino acids that is used to synthesize carnitine. In the carnitine biosynthesis pathway, betaine takes the form of butyrobetaine to synthesize L-carnitine [14]. As a result, it is possible that the decrease in glutaroyl carnitine and betaine could have caused free carnitine levels to decrease in CPT1c KO mice. Previous studies also tested hypothalamic and cortical explants from WT and CPT1c KO mice for their ability to oxidize fatty acids, but there was no evidence that unique properties in neurons existed to allow activation of fatty acid oxidation by CPT1c [5]. CPT1c over-expressed in heterologous cells in vitro also did not show a change in fatty acid oxidation [5]. Therefore, our results remain consistent with previous findings that CPT1c, although it is highly homologous with its isoforms CPT1a and CPT1b, does not participate substantially in neuronal mitochondrial fatty acid oxidation.

Loss of CPT1c results in decreased levels of endogenous endocannabinoids

Several studies have investigated the neurological role of endocannabinoids on food intake [15]. A study investigated the role of endocannabinoids in regulating food intake in the tongue, gut and different brain regions, suggesting that the cannabinoid system plays a role in modulating the activity of neural pathways that regulate food intake and energy expenditure [15]. The brain cannabinoid system, as shown in Figure 3B, regulates food intake through the interaction of endogenous ligands and cannabinoid receptors. From our metabolomic analyses, there was a significant decrease in palmitoylethanolamine and a trend for a decrease in 2-oleolylglycerol in CPT1c KO mouse brains compared to WT mouse brains (Figure 3). There was no significant difference between WT and CPT1c KO mice for free nonesterified fatty acids (Table 1). Among the metabolites shown in Figure 3A, eicosapentaenoate and palmitoylethanolamine showed a significant decrease in CPT1c KO mice with a p-value of 0.0236 and 0.0331, respectively. There was also a slight increase in ethanolamine between WT and CPT1c KO mice, and decrease in 2-oleoylglycerol (p=0.0769), an endogenous cannabinoid (CB) CB-1 agonist (Figure 3A).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_Article_396_Fig3_HTML.jpg
Figure 3

Loss of CPT1c results in decreased endocannabinoids in the brain. (A) Biochemicals involved in fatty acid biochemistry from WT and CPT1c KO mouse brains were compared to determine if metabolomic analyses showed any statistically significant changes. There was an overall decreasing trend in endocannabinoids in CPT1c KO mice. Specifically, eicosapentaenoate (p=0.0236) and palmitoylethanolamine (p=0.0331) significantly decreased in CPT1c KO mice. (B) A schematic of how a decrease in endocannabinoids can induce a decrease in food intake by interacting with CB1 and CB2 cannabinoid receptors.

Loss of CPT1c results in increased levels of glutathione

The oxidized form of GSH (GSSG) and 5-oxoproline, biochemicals involved in the gamma-glutamyl redox cycle, resulted in a statistically significant difference in CPT1c KO mice (Table 2). GSSG and cysteine-glutathione disulfide levels increased while 5-oxoproline levels decreased in CPT1c KO mice (Figure 4A). Based on the schematic redox pathway shown in Figure 4B, our results suggest that CPT1c may play a role in oxidative metabolism. This is consistent with findings in cancer metabolism. Zaugg et al. depleted the levels of CPT1c in MCF-7 cells to determine whether these cells were sensitive to oxidative stress. Hypoxia was used as a stress inducer, and they found that CPT1c depletion caused an increased sensitivity to oxidative stress, implying that CPT1c may play a crucial role in protecting the cells from stress from the environment [13]. Furthermore, the loss of CPT1c resulted in an increase in ceramides [7, 8], a key mediator of oxidative stress [16, 17]. However, the mechanism and role of CPT1c in oxidative metabolism remains unknown.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_Article_396_Fig4_HTML.jpg
Figure 4

Loss of CPT1c results in elevated oxidative demands in the brain. (A) In a comparison of biochemicals involved in redox homeostasis in WT and CPT1c KO mouse brains, GSSG and 5-oxoproline were statistically significant. GSSG levels increased in CPT1c KO mice with a p-value of 0.0307, while 5-oxoproline decreased in KO mice (p=0.0291). The biochemicals shown displayed an overall increasing trend in CPT1c KO mice. (B) A schematic of the gamma-glutamyl redox cycle. Based on the pathway, an increase in the biochemicals from Figure 4A may cause the cells to become more sensitive to oxidative stress.

Discussion

Role of CPT1c in behavior and physiology

Carnitine acyltransferases are enzymes that catalyze the exchange of acyl groups between carnitine and CoA to facilitate the transport of acyl groups from the cytoplasm to the mitochondrial matrix. Carnitine acetyltransferase (CRAT) and carnitine octonyltransferase (CROT) facilitate transport short- and medium-chain acyl-CoA, while CPT1 facilitate transports long chain acyl-CoA to the mitochondria. CPT1 enzymes are encoded by three genes in mammals that are localized in different tissues and have different properties. CPT1a, which is enriched in the liver, has been heavily studied due to its crucial role in β-oxidation and human fatty oxidation disorders (OMIM #255120) and is lethal when knocked out in mice [18]. CPT1b is localized mainly in the muscle and is a regulator for the use of fatty acids in muscle and is also lethal when knocked out in mice [19]. These two enzymes, which are present on the outer mitochondrial membrane, play a critical role in regulating and facilitating fatty acid beta-oxidation.

The brain specific CPT1c is highly homologous to its closely related genes, CPT1a and CPT1b [4]. However, despite its high homology, CPT1c does not catalyze acyl transfer from long chain acyl-CoA to carnitine [46]. Other distinguishing properties of CPT1c include a longer C-terminus and localization in the endoplasmic reticulum (ER) instead of the mitochondria [11]. Although it does not facilitate acyl transfer in the cell, CPT1c most likely remains sensitive to the endogenous allosteric CPT1 inhibitor, malonyl-CoA, binding with a similar affinity as CPT1a [4, 6]. Moreover, while other isoenzymes are expressed in a broad range of organisms, CPT1c seems to have risen late in evolution, raising the question whether CPT1c has a specific role in mammalian brain function.

Several studies used CPT1c knockout (KO) and CPT1c transgenic mice to investigate the role of CPT1c in the CNS. Knockout studies showed that loss of CPT1c did not affect the viability or fertility of the mice, but resulted in a suppression in food intake and decrease in body weight when they were fed a normal or low-fat diet [6, 9]. Paradoxically, when high fat diet was given to CPT1c KO mice, they exhibited diet-induced obesity which ultimately resulted in a diabetic phenotype [5, 6]. Even though fatty acid oxidative metabolites showed no significant change based on the metabolomic analysis, due to a decrease in peripheral energy expenditure CPT1c KO mice were more susceptible to obesity and diabetes when fed a high fat diet. This suggests that CPT1c has a hypothalamic function in protecting the body from adverse weight gain when the mice were fed a high fat diet. Transgenic CPT1c mice (CPT1c-TgN), on the other hand, which allowed conditional expression of CPT1c in a tissue-specific manner via cre-lox recombination, showed enhanced expression of CPT1c and they were protected from diet-induced obesity even on a high-fat diet [10].

CPT1c KO mice also showed impaired spatial learning [7]. Cpt1c deficiency was shown to alter dendritic spine morphology by increasing immature filopodia and reducing mature mushroom and stubby spines. Compared to WT mice, CPT1c KO mice showed a higher escape latency, implying that they had a delay in the acquisition phase [7]. Based on this study, CPT1c deficiency interfered with consolidating new information but did not affect retaining information or motor behavior. As a result, there may be other physiological roles of CPT1c in addition to regulating food intake and energy expenditure consistent with its broad expression throughout the nervous system [7].

Endocannabinoid regulation of food intake

Endocannabinoids are endogenous ligands that bind to cannabinoid receptors to regulate many aspects of physiology and behavior. Specifically, the brain endocannabinoid system regulates food intake via the hypothalamus, where it activates necessary mediators to induce appetite after a short-term food deprivation. CB1 receptor KO mice showed reduced food intake, similar to CPT1c KO mice [20, 21]. Based on our results, CPT1c could be interacting with the cannabinoid system, causing an overall decreasing trend in endocannabinoids in CPT1c KO mice. In this context, the loss of CPT1c could have influenced the endocannabinoid system and its function to regulate food intake and body weight, which may explain the suppressed food intake in CPT1c KO mice [5, 9]. Therefore, a decrease in endocannabinoids based on metabolomic profiling may suggest a putative role of the endocannabinoid system in suppressing food intake in CPT1c KO mice. However, it is unclear if CPT1c affects endocannabinoid metabolism directly or more likely indirectly by altering neuronal specific fatty acid metabolism.

Glutathione and redox metabolism

Neurons are particularly sensitive to oxidative stress and damage caused by reactive oxygen species (ROS). On the cellular level, there are many endogenous metabolic stress inducers, such as ROS produced from the mitochondria and cytosolic enzymes, such as cyclooxygenase and lipoxygenase. There are also various exogenous conditions that can also promote the level of ROS species to increase, such as H2O2 and hypoxia, that induces irreversible cellular damage or cell death. As shown by the pathway in Figure 4B, reduced glutathione (GSH) and oxidized glutathione (GSSG) are tightly regulated in order to maintain cellular redox homeostasis and to protect the cells from oxidative damage [17]. Carrasco et al. showed that CPT1c expression correlated with ceramide production and loss of CPT1c resulted in reduced ceramide levels. [7]. A recent study on the role of CPT1c in cancer cells in response to metabolic stress showed that CPT1c could participate in protecting cells from stress. In addition, they postulated that metabolic stress could alter regulation of the CPT1c gene, reducing ATP production and increasing sensitivity towards metabolic stress [13]. Here, we showed that CPT1c deficiency results in an increased oxidative environment. This may indicate that although CPT1c does not contribute in large part to beta-oxidation, it may be involved in other neuron specific oxidative metabolism. Alternatively, CPT1c may need to be activated in a yet to identified stress-induced manner. Barger et al. [22] showed that CPT1c was required for leukemia growth under low glucose conditions. Therefore, CPT1c may have a context dependent role in fatty acid catabolism. Although here we show that CPT1c could play a role in oxidative stress, the precise role of CPT1c in relation to oxidative stress remains unknown.

Conclusion

Unbiased metabolomic profiling of steady-state metabolites in WT and CPT1c KO brains revealed subtle changes in a broad range of metabolites in vivo. The metabolic alterations are not consistent with CPT1c playing a role in beta-oxidation or a large non-redundant role in bioenergetics.

Abbreviations

WT: 

Wild-type

KO: 

Knockout

CPT1: 

Carnitine Palmitoyltransferase 1

CPT2: 

Carnitine Palmitoyltransferase 2

CoA: 

Coenzyme A

CB: 

Cannabinoids

GC: 

Gas chromatography

MS: 

Mass spectrometry.

Declarations

Acknowledgments

We would like to thank Amanda Reamy for technical assistance. This work was supported in part by the American Heart Association (SDG2310008 to M.J.W.) and NIH NINDS (NS072241 to M.J.W.).

Authors’ Affiliations

(1)
Department of Biological Chemistry, Center for Metabolism and Obesity Research, Johns Hopkins University School of Medicine

References

  1. Cahill GF: Fuel metabolism in starvation. Annu Rev Nutr. 2006, 26: 1-22. 10.1146/annurev.nutr.26.061505.111258.PubMedView Article
  2. Cahoy JD, Emery B, Kaushal A, Foo LC, Zamanian JL, Christopherson KS, Xing Y, Lubischer JL, Krieg PA, Krupenko SA: A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci. 2008, 28 (1): 264-278. 10.1523/JNEUROSCI.4178-07.2008.PubMedView Article
  3. Wolfgang MJ, Lane MD: The role of hypothalamic malonyl-CoA in energy homeostasis. J Biol Chem. 2006, 281 (49): 37265-37269. 10.1074/jbc.R600016200.PubMedView Article
  4. Price N, van der Leij F, Jackson V, Corstorphine C, Thomson R, Sorensen A, Zammit V: A novel brain-expressed protein related to carnitine palmitoyltransferase I. Genomics. 2002, 80 (4): 433-442. 10.1006/geno.2002.6845.PubMedView Article
  5. Wolfgang MJ, Cha SH, Millington DS, Cline G, Shulman GI, Suwa A, Asaumi M, Kurama T, Shimokawa T, Lane MD: Brain-specific carnitine palmitoyl-transferase-1c: role in CNS fatty acid metabolism, food intake, and body weight. J Neurochem. 2008, 105 (4): 1550-1559. 10.1111/j.1471-4159.2008.05255.x.PubMedPubMed CentralView Article
  6. Wolfgang MJ, Kurama T, Dai Y, Suwa A, Asaumi M, Matsumoto S, Cha SH, Shimokawa T, Lane MD: The brain-specific carnitine palmitoyltransferase-1c regulates energy homeostasis. Proc Natl Acad Sci USA. 2006, 103 (19): 7282-7287. 10.1073/pnas.0602205103.PubMedPubMed CentralView Article
  7. Carrasco P, Sahun I, McDonald J, Ramirez S, Jacas J, Gratacos E, Sierra AY, Serra D, Herrero L, Acker-Palmer A: Ceramide levels regulated by carnitine palmitoyltransferase 1C control dendritic spine maturation and cognition. J Biol Chem. 2012, 287 (25): 21224-21232. 10.1074/jbc.M111.337493.PubMedPubMed CentralView Article
  8. Gao S, Zhu G, Gao X, Wu D, Carrasco P, Casals N, Hegardt FG, Moran TH, Lopaschuk GD: Important roles of brain-specific carnitine palmitoyltransferase and ceramide metabolism in leptin hypothalamic control of feeding. Proc Natl Acad Sci USA. 2011, 108 (23): 9691-9696. 10.1073/pnas.1103267108.PubMedPubMed CentralView Article
  9. Gao XF, Chen W, Kong XP, Xu AM, Wang ZG, Sweeney G, Wu D: Enhanced susceptibility of Cpt1c knockout mice to glucose intolerance induced by a high-fat diet involves elevated hepatic gluconeogenesis and decreased skeletal muscle glucose uptake. Diabetologia. 2009, 52 (5): 912-920. 10.1007/s00125-009-1284-0.PubMedView Article
  10. Reamy AA, Wolfgang MJ: Carnitine palmitoyltransferase-1c gain-of-function in the brain results in postnatal microencephaly. J Neurochem. 2011, 118 (3): 388-398. 10.1111/j.1471-4159.2011.07312.x.PubMedView Article
  11. Sierra AY, Gratacos E, Carrasco P, Clotet J, Urena J, Serra D, Asins G, Hegardt FG, Casals N: CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity. J Biol Chem. 2008, 283 (11): 6878-6885. 10.1074/jbc.M707965200.PubMedView Article
  12. Eckel-Mahan KL, Patel VR, Mohney RP, Vignola KS, Baldi P, Sassone-Corsi P: Coordination of the transcriptome and metabolome by the circadian clock. Proc Natl Acad Sci USA. 2012, 109 (14): 5541-5546. 10.1073/pnas.1118726109.PubMedPubMed CentralView Article
  13. Zaugg K, Yao Y, Reilly PT, Kannan K, Kiarash R, Mason J, Huang P, Sawyer SK, Fuerth B, Faubert B: Carnitine palmitoyltransferase 1C promotes cell survival and tumor growth under conditions of metabolic stress. Genes Dev. 2011, 25 (10): 1041-1051. 10.1101/gad.1987211.PubMedPubMed CentralView Article
  14. Sharma S, Black SM: Carnitine homeostasis, mitochondrial function, and cardiovascular disease. Drug Discov Today Dis Mech. 2009, 6 (1–4): e31-e39.PubMedPubMed CentralView Article
  15. Dipatrizio NV, Piomelli D: The thrifty lipids: endocannabinoids and the neural control of energy conservation. Trends Neurosci. 2012, 35 (7): 403-411. 10.1016/j.tins.2012.04.006.PubMedPubMed CentralView Article
  16. Sanvicens N, Cotter TG: Ceramide is the key mediator of oxidative stress-induced apoptosis in retinal photoreceptor cells. J Neurochem. 2006, 98 (5): 1432-1444. 10.1111/j.1471-4159.2006.03977.x.PubMedView Article
  17. Andrieu-Abadie N, Gouaze V, Salvayre R, Levade T: Ceramide in apoptosis signaling: relationship with oxidative stress. Free Radic Biol Med. 2001, 31 (6): 717-728. 10.1016/S0891-5849(01)00655-4.PubMedView Article
  18. Nyman LR, Cox KB, Hoppel CL, Kerner J, Barnoski BL, Hamm DA, Tian L, Schoeb TR, Wood PA: Homozygous carnitine palmitoyltransferase 1a (liver isoform) deficiency is lethal in the mouse. Mol Genet Metab. 2005, 86 (1–2): 179-187.PubMedView Article
  19. Ji S, You Y, Kerner J, Hoppel CL, Schoeb TR, Chick WS, Hamm DA, Sharer JD, Wood PA: Homozygous carnitine palmitoyltransferase 1b (muscle isoform) deficiency is lethal in the mouse. Mol Genet Metab. 2008, 93 (3): 314-322. 10.1016/j.ymgme.2007.10.006.PubMedPubMed CentralView Article
  20. Cardinal P, Bellocchio L, Clark S, Cannich A, Klugmann M, Lutz B, Marsicano G, Cota D: Hypothalamic CB1 cannabinoid receptors regulate energy balance in mice. Endocrinology. 2012, 153 (9): 4136-4143. 10.1210/en.2012-1405.PubMedView Article
  21. Cota D, Marsicano G, Tschop M, Grubler Y, Flachskamm C, Schubert M, Auer D, Yassouridis A, Thone-Reineke C, Ortmann S: The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis. J Clin Invest. 2003, 112 (3): 423-431.PubMedPubMed CentralView Article
  22. Barger JF, Gallo CA, Tandon P, Liu H, Sullivan A, Grimes HL, Plas DR: S6K1 determines the metabolic requirements for BCR-ABL survival. Oncogene. 2012, 10.1038/onc.2012.70. [Epub ahead of print]

Copyright

© Lee and Wolfgang; licensee BioMed Central Ltd. 2012

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