LL--Carnitine: a nutritional Carnitine: a nutritional modulator of the modulator of the glucocorticoidglucocorticoid

receptorreceptor

Salvatore Alesci, M.D., Ph.D.Salvatore Alesci, M.D., Ph.D.

CNE/NIMH & PREB/NICHD CNE/NIMH & PREB/NICHD

National Institutes of HealthNational Institutes of Health

Bethesda, March 26, 2004Bethesda, March 26, 2004

GlucocorticoidsGlucocorticoids

Steroid hormones secreted by the adrenal gland basally, and incrSteroid hormones secreted by the adrenal gland basally, and increasingly in easingly in response to stress response to stress

Key regulators of carbohydrate, lipid and protein metabolismsKey regulators of carbohydrate, lipid and protein metabolisms

Potent antiPotent anti--inflammatory and inflammatory and immunomodulatoryimmunomodulatory effectseffects

Essential for the normal functioning of the central nervous systEssential for the normal functioning of the central nervous system, and the em, and the maintenance of cardiovascular, metabolic and immune maintenance of cardiovascular, metabolic and immune homeostaseshomeostases

Treatment of choice for many inflammatory, autoimmune and Treatment of choice for many inflammatory, autoimmune and lymphoproliferativelymphoproliferative disordersdisorders

Physiologic and pharmacologic actions mediated by intracellular Physiologic and pharmacologic actions mediated by intracellular receptors,receptors,the the glucocorticoidglucocorticoid receptors (receptors (GRsGRs) )

Human Human GlucocorticoidGlucocorticoid ReceptorReceptorTGA, ßTGA, ßTGA, aTGA, aATGATG

11 22 33 44 55 66 77 88 99αα 99ββ

Chromosome 5Chromosome 5

GRGRαα cDNAcDNA 11 22 33 44 55 66 77 88 99αα

99ββ11 22 33 44 55 66 77 88GRGRββ cDNAcDNA

Immunogenic DomainImmunogenic Domain DBDDBD LBDLBD11 777777

GRGRαα HRHR

GREsGREs

RAPsRAPs

GRGRNucleusNucleus

Cell MembraneCell Membrane

AssemblyAssembly

TFETFE

LigandLigand

ExportExport

LigandLigand BindingBinding

ImportImport

TFTF

GlucocorticoidGlucocorticoid--like effects of Llike effects of L--CarnitineCarnitine

Suppression of LPSSuppression of LPS--induced cytokine productioninduced cytokine production in rodentsin rodents(Med. (Med. InflammInflamm. 2:S43. 2:S43--50, 1993; Br. J Cancer 72:117350, 1993; Br. J Cancer 72:1173--79, 1995)79, 1995)

Reduced Reduced ex vivo ex vivo release of release of TNFTNFαα by by S. S. aureusaureus--stimulated stimulated human human PMNCsPMNCs(Med. (Med. InflammInflamm. 2:S37. 2:S37--41, 1993) 41, 1993)

Decreased serum Decreased serum TNFTNFαα levelslevels in surgical and AIDS patients in surgical and AIDS patients (Med. (Med. InflammInflamm. 2:S33. 2:S33--36, 1993; 36, 1993; ImmunopharmacolImmunopharmacol. . ImmunotoxicolImmunotoxicol. 15:1. 15:1--12, 1993)12, 1993)

Equal efficacy of LEqual efficacy of L--CarnitineCarnitine and and betamethasonebetamethasone administered to pregnant administered to pregnant ratsrats in increasing the in increasing the dipalmitoyldipalmitoyl--phosphatidylcholinephosphatidylcholine content of the fetal content of the fetal lunglung

((PediatrPediatr. Res. 18:1246. Res. 18:1246--52, 1984; J. 52, 1984; J. PerinatPerinat. Med. 24:591. Med. 24:591--99, 1996)99, 1996)

HypothesisHypothesis

The The GlucocorticoidGlucocorticoid--like effects of high like effects of high LL--CarnitineCarnitine doses may be mediated doses may be mediated via direct modulation of via direct modulation of GRGRαα functionfunction

ObjectiveObjectiveTo study To study in vitro in vitro the effect of Lthe effect of L--CarnitineCarnitine on: on:

GRGRαα

Binding capacityBinding capacity

Cell traffickingCell trafficking

Transcriptional activityTranscriptional activity

Biological activityBiological activity

Competitive BindingCompetitive Binding

LL--Carnitine (Carnitine (mMmM))

SWC

B o

f SW

CB

of 33 H

H D

exDex

(% o

f Con

trol

)(%

of C

ontr

ol)

00

2020

4040

6060

8080

100100

120120

00 1010 2020 3030 4040 5050 6060 7070 8080 9090 100100

HeLaHeLa

**

**** ************

Saturation BindingSaturation Binding

No Car: B max = 202.1 +/- 6.46 Kd

= 8 +/- 0.633 25 mM Car: B max = 201.7 +/- 4.46 K

d= 11.8 +/- 0.696

50 mM Car: B max = 180.5 +/- 7.72 Kd = 14.0 +/- 0.919 100 mM Car: B max = 183.2 +/- 11.9 Kd = 23.0 +/- 2.100

No CarNo Car25 25 mMmM CarCar50 50 mMmM CarCar

100 100 mMmM CarCar

200200

002020404060608080

100100120120140140160160180180

00 2020 4040 6060

DexamethasoneDexamethasone ((nMnM))

SWC

B (f

mol

es/1

0SW

CB

(fm

oles

/106 6

cells

)ce

lls)

HeLaHeLa

00

55

1010

1515

2020

2525

00 5050 100100 150150 200200

SWCB (fmoles/10SWCB (fmoles/1066 cells)cells)B

ound

/Fre

eB

ound

/Fre

e

GRGRαα--GFP translocationGFP translocation15 min15 min 30 min30 min00

DexDex(10(10--66 M)M)

LL--CarCar(50 (50 mMmM))

Reporter PlasmidsReporter Plasmids

MMTV:LUCMMTV:LUC

GREGRE GREGRE GREGRE LUCLUCTATATATAGREGRE

TATATATA ββ−−GalGalSV40:SV40:ββ--GalGal

Promoter transcriptionPromoter transcription

0.00.0

0.50.5

1.01.0

1.51.5

2.02.0

2.52.5

L-Car(100mM)

Dex(10-8 M)

RU 486RU 486(10(10--66 M)M)

--

--

--

++

--

--

++

--

--++

--

-- ++

--

++

1010 2525 5050 100100

---- -- --DexDex(10(10--88 M)M)

Cor

rect

ed

Cor

rect

ed L

ucife

rase

Luci

fera

seA

ctiv

ityA

ctiv

ity

HeLaHeLa: MMTV: MMTV

LL--CarCar((mMmM))

0000

++--

00

0.50.5

1.01.0

1.51.5

2.02.02.52.5

3.03.0

3.53.5

Reporter PlasmidsReporter Plasmids

TAT3:LUCTAT3:LUC

GREGRE GREGRE GREGRE ADHADH LUCLUCTATATATA

ADHADH LUCLUCTATATATApODLO2:LUCODLO2:LUC

TATATATA ββ−−GalGalSV40:SV40:ββ--GalGal

Promoter transcriptionPromoter transcription

00

22

44

66

88

1010

1212

Cor

rect

ed

Cor

rect

ed L

ucife

rase

Luci

fera

seA

ctiv

ityA

ctiv

ity

LL--CarCar((mMmM))

DexDex(10(10--88 M)M)

0000

++--

HeLaHeLaTAT3TAT3

1010

--

2525 5050 100100

-- -- --

22

44

66

88

1010

1212

00

0000

++--

1010

--

2525 5050 100100

-- -- --

ODLO 2ODLO 2

Promoter transcriptionPromoter transcription

0.00.00.50.51.01.0

1.51.5

2.02.02.52.53.03.0

3.53.5hGRhGRαα ++

--

--

--++

--

-- ++

--

-- 2525 5050 100100

-- -- --

-- -- -- ++

--

++

2525 5050 100100

-- -- --

++ ++ ++

CVCV--1: MMTV1: MMTV

--

--

--++

--

-- ++

--

-- 2525 5050 100100

-- -- --

-- -- -- ++

++

2525 5050 100100

-- -- --

++ ++ ++

--0.00.00.50.51.01.0

1.51.5

2.02.02.52.53.03.0

3.53.5hGRhGRαα --

LL--CarCar((mMmM))

DexDex(10(10--88 M)M)

RU 486RU 486(10(10--66 M)M)

Cor

rect

ed

Cor

rect

ed L

ucife

rase

Luci

fera

seA

ctiv

ityA

ctiv

ity

CytotoxicityCytotoxicity assayassay

XTTXTT

XTT Formazan

492nM

18 h18 h

24 h24 h

CytotoxicityCytotoxicity assayassay

00

0.50.5

11

1.51.5

22

2.52.5

33

3.53.5

00 100100 200200 300300 400400 500500

HeLaHeLa

LL--Carnitine (Carnitine (mMmM))

Abs

orba

nce

Abs

orba

nce

(A(A49

2 nm

492

nm-- AA

690

nm69

0 nm

))

LPSLPS--stimulated cytokine secretionstimulated cytokine secretion

++

-- 5050 100100

-- --

++ ++

-- ++

--

++ ++

-- 5050 100100

-- --

++ ++

-- ++

--

++

Human elutriated Human elutriated monocytesmonocytes

00

2020

4040

6060

8080

100100

120120

140140

TNF

TNFαα

(% o

f con

trol

)(%

of c

ontr

ol)

LL--CarCar((mMmM))

DexDex(10(10--88 M)M)

RU 486RU 486(10(10--66 M)M)

--

--

--

--

--

--

00

2020

4040

6060

8080

100100

120120

140140

ILIL-- 1

2 (%

of c

ontr

ol)

12 (%

of c

ontr

ol)

****

++

--

--

5050 100100

-- --

-- --

******

++

--

--

5050 100100

-- --

-- --

****

******

SummarySummaryIn vitroIn vitro, high but non, high but non--cytotoxiccytotoxic concentrationsconcentrations of Lof L--CarnitineCarnitineare able to:are able to:

Compete with Compete with dexamethasonedexamethasone for binding to the for binding to the GRGRαα, , reducing the affinity of this receptor for its native reducing the affinity of this receptor for its native ligandligand

Trigger nuclear translocation of Trigger nuclear translocation of GRGRαα

Stimulate the transcription of Stimulate the transcription of glucocorticoidglucocorticoid--responsive responsive promoters through promoters through GRGRαα transactivationtransactivation

Suppress the release of Suppress the release of TNFTNFαα and ILand IL--12 from human 12 from human elutriated elutriated monocytesmonocytes in a in a GRGRαα--dependent fashiondependent fashion

Proposed model of GRProposed model of GR modulation by Lmodulation by L--CarnitineCarnitine

Inactive TDInactive TD

RAPsRAPs

Active TDActive TD

Nuclear translocationNuclear translocation

Transcriptional activationTranscriptional activation

GlucocorticoidGlucocorticoid

Decreased affinity Decreased affinity for native for native ligandligand

Inactive TDInactive TD

LL--CarnitineCarnitine Nuclear translocationNuclear translocation

Transcriptional activationTranscriptional activationActive TDActive TD

Future ResearchFuture ResearchMolecular and structural mechanisms of LMolecular and structural mechanisms of L--CarnitineCarnitine GRGRαα interactioninteraction

Tissue specificity/selectivity of Tissue specificity/selectivity of GRGRαα modulation by Lmodulation by L--CarnitineCarnitine (muscle, (muscle, bone, adipose tissue, neurons)bone, adipose tissue, neurons)

Effect of LEffect of L--CarnitineCarnitine metabolites (Acetylmetabolites (Acetyl--LL--CarnitineCarnitine, , PropionylPropionyl--LL--CarnitineCarnitine, etc.) on , etc.) on GRGRαα function function

Effect of LEffect of L--CarnitineCarnitine and its metabolites on the activity of other steroid and its metabolites on the activity of other steroid receptors (PR, MR, AR, etc.) receptors (PR, MR, AR, etc.)

Studies in animal models of chronic inflammatory and autoimmuneStudies in animal models of chronic inflammatory and autoimmunedisordersdisorders

Clinical trialsClinical trials

Drug designing Drug designing

AcknowledgementsAcknowledgements

Salvatore BenvengaSalvatore BenvengaFrancesco Trimarchi

George P. ChrousosGeorge P. ChrousosTomoshige KinoTomoshige Kino

Massimo U. De MartinoMassimo U. De MartinoMarco Marco Mirani

Francesco Trimarchi

Mirani

PieroPiero OrtecaOrteca

Philip W. GoldPhilip W. Gold AntoninoAntonino AmatoAmato