Dolors Serra Departament de Bioquímica i Biología Molecular

Noves estrategies de

modulació del metabolisme lipídic

per al tractament de l’obesitat

Seminaris de Recerca de la Facultat de Farmàcia

8 de Maig de 2012

2

Insulin resistance

and Diabetes

Cancer

Cardiovascular

disease

Mechanical

stress

Obesity

Food intake Energy expenditure

• Currently, 1.600 million

adults worldwide are

overweight and 312 million

of them are clinically obese

• Obesity is also a serious

health concern for children

17% obese children (US)

Prevalence and trends

BMI: kg/m2

BMI>25 overweight

BMI>30 obese

Obesity in the World 2012

Economic impact-2010

• Obesity is a multi-level problem and

requires a multi-level approach to integrate

treatment and prevention:

- Individual and Family

- School and Community

- Government

Obesity Interventions

Policies focused on changing behaviours

Healthy diet Increasing physical activity

Policies focused on changing behaviours

Anti-obesity Drugs

2012

New strategies

EL PAIS

Irisina, hormona quemagrasas

Irisina, hormona contra la obesidad

Recientes investigaciones sacaron a la luz un nuevo descubrimiento, una hormona quemagrasas

llamada Irisina. Esta hormona es sumamente importante para el tratamiento contra la obesidad. Al

parecer cuando realizas actividad física, se ponen en marcha una serie de procesos metabólicos que

favorecen la síntesis de la hormona Irisina, que tendría efectos directos sobre el tejido graso.

LA VANGUARDIA.com/salud

Irisina, la hormona de la actividad física

Científicos de Harvard descubren una sustancia clave en los efectos saludables del deporte y buscan un

fármaco que imite su actividad | Empleada como fármaco, podría mejorar el tratamiento de la diabetes y

la obesidad

Salud | 13/01/2012 - 02:44h

Ana Macpherson Barcelona , Josep Corbella Barcelona

Pathophysiology of Obesity

Lewis et al. Endocrine Reviews, 23, 2002

Estratègies contra l’obesitat

• Augment de l’oxidació d’àcids

grassos en diferents teixits

• Control de la ingesta a nivel del SNC

Carnitina

Palmitoiltransferasa 1

Ácidos grasos

β-oxidación

ATP

Mitocondria

Oxidación de los ácidos grasos

Ácidos grasos

CPT1 Malonil-CoA

CPT1M

Dr. Paulino Gómez Puertas

CBM Severo Ochoa CSIC-UAM ( Madrid)

Viral vectors

Ad-LacZ

Ad-CPT1

Ad-CPT1M

1) Expresión a largo plazo

2) No respuesta inmune

Adenovirus

Virus Adenoasociados

AAV1-GFP

AAV1-CPT1A

AAV1-CPT1AM

Amsterdam Molecular Therapeutics

Netherlands

Insulin

PKB

Glucose

Glucose

FFA

LCACoA

DAG/Ceramide

-oxidation

CPT I

Insulin

PKB

Glucose

Glucose

FFA

LCACoA

DAG/Ceramide

-oxidation CPTI

CPT I CPTI

Reversión de la resistencia a la insulina inducida por palmitato

Células musculares L6E9

Ad-LacZ Ad-LCPT Im Ad-LCPT Iwt

88 kDa

CP

T I A

cti

vit

y

(nm

ol x

min

-1 x

mg

pro

t-1)

control etomoxir

Ad-LacZ Ad-LCPT Iwt Ad-LCPT Im

** *

0

2,5

5,0 7,5

10,0 12,5 15,0 17,5

20,0

Sebastián D. et al. AJP. 2007

Pa

lmit

ate

ox

ida

tio

n t

o C

O2

(fo

ld in

cre

as

e)

*

*

*

#

# #

#

0

0,5

1

1,5

2

2,5 2,5 mM Glc

15 mM Glc

25 mM Glc

Ad-LacZ Ad-LCPT I

wt

Ad-LCPT I

M593S

#

#

Sobreexpresión de la CPT1A en células musculares L6E9

Sebastián D. et al. J Lipid Res. 2009

Ad-LacZ

Ad-CPT1

Ad-CPT1M

Insulin

PKB

Glucose

Glucose

FFA

LCACoA

DAG/Ceramide

-oxidation

CPT I

Insulin

PKB

Glucose

Glucose

FFA

LCACoA

DAG/Ceramide

-oxidation

LCPTI

M593S

CPT I LCPTI

M593S

La sobreexpresión de CPT1 en células musculares mejora la resistencia a la insulina inducida por palmitato

Sebastián D. et al. AJP. 2007

Sebastián D. et al. J Lipid Res. 2009

Our strategy in the liver

-oxidation acetyl-CoA

ECT

CO2 ketone bodies

TCA

CPT1A

acyl-CoA 1. Overexpression of a CPT1AM isoform insensitive to malonyl-CoA.

2. Long term overexpression using as a vector adenoassociate virus (AAVs).

CPT1AM

Hepatocyte

ATP

AAV1-GFP

AAV1-CPT1A

AAV1-CPT1AM

Two animal models: Obesity induced by HFD

Genetic obesity

Effect of CPT1AM overexpression on HFD Mice

Mice C57BL/6

ControlDiet

High Fat Diet

AAV-GFP

AAV-CPT1AM

AAV-CPT1A

AAV-GFP

AAV-CPT1AM

AAV-CPT1A

NCD

HFD

10

Injection

AAV

23 8 14

Age

(weeks)

Sacrifice Sacrifice

10

AAV

23 8 14

NCD o HFD

CPT1 Activity

GFP CPT1A CPT1AM

Activity C

PT

1

( nm

ol /m

g p

rot .m

in)

1

2

3

4

5 * *

( nm

ol /m

g p

rot .m

in)

1

2

3

4

5

NCD

Activity C

PT

1

( nm

ol /m

g p

rot .m

in) *

1

2

3

4

5

GFP CPT1A CPT1AM 0

*

( nm

ol /m

g p

rot .m

in)

1

2

3

4

5

0

HFD

CPT1A and CPT1AM liver activity

To

tal O

lea

te o

xid

atio

n

’

( nm

ol

ole

at /

mg p

rot .

h)

0

20

40

60

GFP CPT1A CPT1AM

NCD

*

* *

( nm

ol

ole

at /

mg p

rot .

h)

0

20

40

60

GFP CPT1A CPT1AM

*

* *

0

20

40

60

GFP CPT1A CPT1AM ( nm

ol

ole

at /

mg p

rot .

h)

HFD

*

* *

0

20

40

60

GFP CPT1A ( nm

ol

ole

at /

mg p

rot .

h)

*

Oleic oxidation

To

tal O

lea

te o

xid

atio

n

Hepatocyte primary culture

HFD GFP

HFD CPT1A

HFD CPT1AM

NCD GFP

NCD CPT1A

NCD CPT1AM

BW

(g)

0

20

25

30

35

40

0 10 14 18 22

Age (weeks)

*

*

* AAV

0

20

25

30

35

40

0 10 14 18 22

*

GFP CPT1A CPT1AM GFP

HFD

Analysis of the phenotipe

Weight

Orellana-Gavaldà et al. Hepatology 2011

0

40

80

120

160

GFP CPT1A CPT1AM

* *

0

40

80

120

160 * *

0

0.2

0.4

0.6

0.8

* *

GFP CPT1A CPT1AM 0

0.2

0.4

0.6

0.8

* *

HFD NCD

Glu

cose

(mg/d

l)

Insulin

(ng/m

l)

11 weeks

Restored glucose and insulin levels

PTT HFD

Glu

co

se

(m

g/d

l)

0

100

200

300

20 40 60 80 100 120

Time (min.)

*

*

0

100

200

300

20 40 60 80 100 120

*

*

HFD CPT1AM

HFD CPT1A HFD GFP

NCD GFP

HFD CPT1AM

HFD CPT1A

GTT HFD

HFD GFP

NCD GFP

Glu

co

se

(m

g/d

l)

0

200

400

600

20 40 60 80 100 120

Time (min.)

*

*

0

200

400

600

20 40 60 80 100 120

*

*

RESULTATS: TAG

LCFA

-oxidation

acetyl-CoA

citrate

acetyl-CoA

malonyl-CoA LC-CoAs TAG

ECT

CO2

ATP Ketone bodies

TCA

Hepatocyte

CPT1A CPT1AM

CPT1A

Lipid metabolism

High Fat Diet

HFD NCD

40X

GFP CPT1A CPT1AM

40X 40X 40X

40X 40X

d

a b c

e f

40X

GFP CPT1A CPT1AM

NC

D

HF

D

40X 40X 40X

40X 40X

20

40

60

80

0 GFP CPT1A CPT1AM

* *

20

40

60

80

TA

G

(m

g/g

fe

tge

)

Decreased steatosis

LCFA

-oxidation

acetyl-CoA

citrate

acetyl-CoA

malonyl-CoA LC-CoAs TAG

DAG

ceramides PKC-ε

Ser/Thr

Fosforilation

IRS-1/IRS-2

PI3K

PDK1

Akt

GSK

glucose

glicogen

ECT

CO2

ATP Ketone bodies

GS

insulin

High Fat Diet

TCA

Hepatocyte

CPT1

ROS

IL- 6

TNFα

Inflamation and oxidative stress

LCFA

CPT1A

-oxidation

acetyl-CoA

citrate

acetyl-CoA

malonyl-CoA LC-CoAs TAG

DAG

ceramides PKC-ε

Ser/Thr

Phosphorilation

IRS-1/IRS-2

PI3K

PDK1

Akt

GSK

glucose

glycogen

ROS

IL- 6

TNFα

ECT

CO2

ATP Ketone bodies

GS

insulin

High Fat Diet

TCA

Hepatocyte

CPT1AM

CPT1A

Effect of CPT1A or CPT1AM expression

Adipocyte

Macrofages Macrofages

Hypertrofy

Leptina,TNFα, IL-6, MCP-I

Effect on Adipose tissue

GFP CPT1A CPT1AM

NCD

HFD

a b

e

c

f

20X

20X 20X 20X

20X 20X

d

20X

20X 20X 20X

20X 20X

GFP CPT1A CPT1AMGFP CPT1A CPT1AM

ROS

Inflamation

Insulin sensitivity normalized

Inflamation, weight, leptin

Prevention

Weight increase

Insulin sensitivity normalized

HFD

Insulin sensitivity normalized CPT1A CPT1AM

CPT1A

β-oxidation

acyl-CoA

TAG

Conclusion

Orellana-Gavaldà et al. Hepatology 2011

db /+ GFP db /+ CPT1AM

db / db GFP

db / db CPT1AM

db /+ GFP db /+ CPT1AM

db / db GFP

db / db CPT1AM

Age (weeks)

AAV-CPT1AM

0

10

20

30

40

50

60

0 5 10 15 20 0 5 10 15 20

BW

(g

)

CPT1AM GFP CPT1AM GFP CPT1AM GFP

Glu

cose (m

g/d

l)

0

100

200

300

db /+ db / db

* *

0

100

200

300

db /+ db / db

* * In

sulin

a (

ng /m

l)

0

2

4

6

8

10

db /+ db / db

* * In

sulin

( n

g /m

l)

0

2

4

6

8

10

db /+ db / db

* *

db/db db/+

Effect of CPT1AM overexpression on genetically obese db/db mice

AAV-GFP

AAV-CPT1AM

c

40X

d

40X

LIVER db/db

40X

40X

CPT1AM

Orellana-Gavaldà et al. Hepatology 2011

2-Control de la ingesta

2-Papel de la CPT1 en el control de la ingesta

Metabolitos

(glucosa, ácidos grasos)

Hormonas

(insulina, leptina)

CONTROL DE LA INGESTA

Citosol Mitocondria

Palmitoil-CoA

Ingesta

β-oxidación

Acetil-CoA Malonil-CoA Palmitato

CPT1

FAS

Aproximación farmacológica

ob/ob mice (leptin deficient)

Diet-induced obese (DIO) mice

Lean mice

control C75

Reduced food intake and dramatic weight loss

Loftus et al. Science. 2000

Acetyl-CoA + Malonyl-CoA FAS

C75

Fatty acids

C75: inhibitor of fatty acid synthase (FAS)

C75 es un inhibidor de la ingesta

Control

Etomoxir

C75 * P 0.05

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

Control C75

Sp

ecific

CP

T1

Activity

(nm

ol/m

in/m

g p

rote

in)

*

0

5

10

15

20

25

1 2 4 22

Time after injection (h)

Cu

mu

lative

Fo

od

In

take

(g

) * *

*

*

-8

-6

-4

-2

0

2

4

Time after injection (22 h)

Bo

dy W

eig

ht

Ch

an

ge

(g

)

*

Mera P.et al. Biochemical Pharmacology (2009)

Efecto del C75 en la actividad CPT1 y en la ingesta

C75 estereotaxis

CPT1

Citosol

Mitocondria

Acetil-CoA

FAS

Palmitoil-CoA

Ingesta

C75 C75-CoA

ACC AMPK

Malonil-CoA Palmitato

Bentebibel A. et al. Biochemistry (2006)

Mera P.et al. Biochemical Pharmacology (2009)

β-oxidación

Kuhajda FP et al. Trends Pharmacol Sci (2005)

Mecanismo de acción del C75 en hipotálamo

CPT1

Hipotálamo

Hígado Páncreas

Músculo

Tejido adiposo

Bentebibel A. et al. Biochemistry (2006)

Mera P.et al. Biochemical Pharmacology (2009)

Gao S. et al. Am J Physiol Regul Integr Comp Physiol (2011)

Gao S. et al Proc Natl Acad Sci U S A. (2011)

Carraco P. et al. J. Biol Chem (2012)

Herrero L. et al. Diabetes (2005)

Orellana-Gavaldà et al. Hepatology (2011)

Sebastián D. et al. Am. J. Physiol .

Endocrinol. Metab. 2007

Sebastián D. et al. J Lipid Res. 2009

Centro de Investigación Biomédica En Red

Fisiopatología de la Obesidad y Nutrición

Centro de Investigación Biomédica En Red

Fisiopatología de la Obesidad y Nutrición

Paulino

REGULATION OF LIPID METABOLISM GROUP (http://www.ub.edu/betaoxi/home.html)

Colaboradores:

Paulino Gómez Puertas

CBM Severo Ochoa CSIC-UAM ( Madrid)

Ana Maria Gómez-Foix

Universidad de Barcelona

AMT (Amsterdam Molecular Therapeutics)

Holanda

Jordi Garcia y Xavier Ariza

Universidad de Barcelona

Núria Casals

Universidad Internacional de Catalunya

Su Gao y Gary Lopaschuck

University of Alberta. Edmonton.Canada