Metabolomic profiling reveals a role for CPT1c in neuronal oxidative metabolism

  • Jieun Lee1 and

    Affiliated with

    • Michael J Wolfgang1Email author

      Affiliated with

      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 KOCPT1c 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 KOCPT1c 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 KOCPT1c 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 KOCPT1c 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.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_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).
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_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).
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_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.
      http://static-content.springer.com/image/art%3A10.1186%2F1471-2091-13-23/MediaObjects/12858_2012_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

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      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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