Doctoral thesis defense Petar Petrov version 5 Feb 2016

CHARACTERIZATION OF THE ROLE OFRETINOBLASTOMA PROTEIN IN METABOLIC REGULATION

Doctoral Thesis Defense

Petar Petrov 5 February 2016, Palma de Mallorca, Spain

pRb

cell cycle control

chromosomal stability

senescencecell

differentiation

apoptosis control of metabolism

The retinoblastoma protein (pRb) is a tumor suppressor protein that is involved in several biological processes

Adipose organ

The adipose organ and the skeletal muscle are a dual hub for energy homeostasis

Liver Brain

satiety

adipokines, myokines Skeletal muscle

Fasting

Energy disposal

Exercise

Cold

Adaptive thermogenesis

Feeding

Energy storage

Adverse metabolic consequences of dysfunctional WAT can be reverted by browning of WAT and activation of BAT and SM

trainingmusclecachexia

lipodystrophy

MetS (insulin resistance, hyperglycemia, dyslipidemia,

hypertension, systemic inflammation)

cold exposure; β-adrenergic stimulation; adrenergic-independent

mechanisms

WAT obesityBAT

activated BAT

browned WAT

White adipocytes

Brown adipocytes

Brite/beige adipocytes

Function Energy storage and release

Adaptivethermogenesis

Adaptive thermogenesis?

Lipid dropletsappearance Unilocular Multilocular Mixed

Mitochondria content Low High Intermediate

(upon stimulation)

Ucp1 protein content

Almost undetectable

High, increases upon stimulation

Increases upon stimulation

Mechanism ofglucose uptake

Insulin-stimulatedGLUT4 translocation

Insulin-stimulatedGLUT4 translocation;norepinephrine -stimulated GLUT1expression andtranslocation

Insulin-independentGlut1 expression andtranslocation

Adipose organ demonstrates metabolic plasticity from WAT to BAT and in between

Skeletal muscle metabolic plasticity

↑ mitochondria quantity↑ oxidative capacity

Slow-twitch myotubes (Type I fibers)

preferentially use fatty acids for ATP production

CO2

glucoseoxidation CO2

WAT NEFA

glucose

small intestine

pancreas

insulin

β-oxidation

Fast-twitch myotubes (Type II fibers)

preferentially use glucose for ATP production

↓ mitochondria quantity↓ oxidative capacity

adapted from Henley and Dick, 2012

phosphorylation by CDKs

dephosphorylation by PPs

hypohosphorylated, active pRB

hyperhosphorylated, less active pRB

pRb

pRb

TF

TF

pRb is a pocket protein whose activity is modulated by post-translational modifications

pRb is a transcriptional co-regulator

activation

MyoD

MyogeninpRb

MyotubesAdipocytes

C/EBPα

pRb

repressionHDACG0/G1

E2F

pRbHDACG1>S

E2F

pRb

pRb

Preadipocytes

Cell cycle

TFPGC1α

promoter

pRb has an important role in adipogenic and myogenic differentiation

(adapted from Park et al., 2014)

pRb

pRb

pRb

pRb?

pRb?

pRb?

Gene Inactivation type Metabolic phenotype References

Rb1 Germ-line Rb1 null Mice die in the early embryonic stages

Jacks et al., 1992; Lee et al., 1992

Rb1Adipose tissue specific Rb1 deletion in adult mice

Resistance to diet-induced obesity due to increased total energy expenditure, activation of brown adipose tissue and conversion of white into brown fat

Dali-Youcef et al., 2007

Rb1Hypothalamus ARC Pomc neurons-specific Rb1 deletion

Pomc-Rb1 floxed mice had higher food intake and % body fat and displayed increased serum leptin, insulin and glucose concentration, accompanied by glucose intolerance

Lu et al., 2013

Rb1Pancreatic β-cell specific Rb1 deletion

No obvious phenotype Vasavada et al., 2007

Rb1, p130

Germ-line double Rb1 and p130 null

Reduced pancreas and β-cell mass; mild hyperglycaemia

Harb et al., 2009

Experimental evidence links pRb to the control of metabolism

Experimental evidence links pRb to the control of metabolism

Rb+/- model

non-obesogenic diet/ageing?acute physiological stress?

General objective:to gain a better and more complete understanding of the role of pRb in the control of metabolism, with a main focus on adipose tissue and skeletal muscle.

Main hypothesis:In normal physiological conditions pRb is involved in metabolic regulation through effects on adipose tissue and other metabolically active tissues such as the skeletal muscle and alteration of adipose pRb status may accompany obesity.

DOCTORAL THESIS “CHARACTERIZATION OF THE ROLE OF RETINOBLASTOMA PROTEIN IN METABOLIC REGULATION”

IV. Do bioactive compounds modulating

adipogenesis affect pRb status?

I. Is pRb involved in human adipogenesis?

II. Is pRb involved in the maintenance of

the mature adipocyte phenotype?

VII. Does pRb have a role in the control of skeletal muscle cell

metabolism?

III. Does obesity affect pRb status in

adipose tissue?

VI. Is the browning potential of WAT- derived

stromal vascular fraction of Rb+/- mice increased?

V. Do Rb+/- mice respond distinctly to metabolic stressors and as they age?

pRb in the control of

metabolism

VIII. Could the Rb+/- mouse model be used for validation of blood

transcript-based biomarkers of health?

Specific objectives

CENIT–PRONAOS

DIABAT

BIOCLAIMS

CIBERobn






VII. Does pRb have a role in the control of skeletal muscle cell metabolism?


adipose tissue?

VI. Is the browning potential of WAT- derived stromal vascular fraction of Rb+/-

mice increased?



metabolism



Specific objectives

CENIT–PRONAOS

DIABAT

BIOCLAIMS

CIBERobn

Experimental design

protein and total RNA isolation, qPCR, immunoblotting

(II) transient silencing of Rb1 in differentiated 3T3-L1 adipocytes

reverse- transfect with siRNA

electroporate with siRNA

replate for 36h

detachsilenced3T3-L1

adipocytes

3T3-L1 adipocytes

confluent 3T3-L1 preadipocytes

differentiate for 7 days

(I) Rb changes during differentiation of human SAT and VAT preadipocytes

day: 0 2 5 7 9 12 14human

adipocytes

confluent human preadipocytes

protein and total RNA isolation, qPCR, immunoblotting, ELISA

pRb activity

RB1 mRNA, protein quantity and activity increased gradually during human preadipocyte differentiation

BMI<25

BMI>30

RB1 mRNA (R.U.)#

#

##

*

*

****

**

** **

####

# ########

#

**

* *

SAT adipocytes

VATadipocytes

SAT adipocytes, BMI<25SAT adipocytes, BMI>30

** P < 0.005 in comparison with day 0; ++ P < 0.005 in comparison with day 7 Day

RB1 total protein (ng/mg total protein)

**++

**

**

0 7 14

phosphoThr821 RB1/ total RB1

0 2 5 7 9 12 14 0 2 5 7 9 12 14 day

*, **: P < 0.05, P < 0.005 in comparison with day 0; #, ##: P < 0.05, P < 0.005 in comparison with day 2

0 7 14 day

siC siRb10

0.00010.00020.00030.00040.00050.00060.0007

*

siC siRb10.000099

0.00009950.0001

0.00010050.000101

0.00010150.000102

0.0001025

siC siRb10

0.00005

0.0001

0.00015

0.0002

siC siRb10

0.0002

0.0004

0.0006

0.0008

*

siC siRb10

0.0005

0.001

0.0015

0.002

*

siC siRb10

0.0020.0040.0060.008

0.010.0120.014

*68 % reduction

UCP1

ADIPOQ

ACTB

siC siRb1siC siRb1

FASN

PPARγ

RB1

Rb1 knockdown led to a loss of the mature adipocyte phenotype m

RNA

expr

essi

on (R

.U)

prot

ein

siRb1

Rb1 Fasn Pparg

Adipoq Pprdm6 Ucp1

A group of 76 men and women with a BMI between

20 and 58 kg/m2

protein and total RNA isolation; qPCR, immunoblotting; correlation analysis with biometric and circulating parameters

Previously collected visceral and subcutaneous

WAT biopsies

Animal experiment

Rats subjected to obesogenic diet with/

without subsequent weight reduction

Previously collected visceral WAT

(retroperitoneal)

Experimental design

Human cohort

A group of 76 men and women with a BMI between

20 and 58 kg/m2

Previously collected visceral and subcutaneous

WAT biopsies

(III) Changes of adipose Rb in obesity

Biobancos-FATBANK

Adipose RB1 mRNA expression was negatively associated with BMI/adiposity

r= -0.332, p= 0.003

RB1 (R.U.) VAT

BMI (kg/m2)

r= -0.269, p= 0.01

RB1 (R.U.) SAT

Human cohort

Rb1 (R.U.) rWAT

Adiposity index

Animal cohort

0

200

400

600

800 *#

* P < 0.05 in comparison with NF; # P < 0.05 in comparison with Cafeteria

r= -0.587, p= 0.003

body weight (g)

BMI (kg/m2

Human adipose RB1 mRNA expression was positively associated with adipogenic gene expression in WAT depots

Correlations of RB1 gene expression (RU) with:VAT (n=76) SAT (n=76)r p r p

Age (years) 0.041 0.718 0.288 0.012Body Mass Index (Kg/m2) -0.332 0.003 -0.269 0.019 Fasting Glucose (mg/dl) -0.221 0.060 -0.023 0.845Fasting Insulin (U/ml) (n=34) -0.471 0.005 -0.446 0.009 HOMAIR (n=34) -0.454 0.007 -0.404 0.018 HDL-Cholesterol (mg/dl) 0.210 0.070 0.032 0.801Fasting Triglycerides (mg/dl) -0.182 0.120 -0.103 0.397Gene expresión (RU)

LEPTIN -0.148 0.337 -0.295 0.010 GLUT4 0.392 0.002 0.192 0.187

IRS1 0.358 0.004 0.413 0.001 PPARγ 0.414 0.001 0.460 <0.0001

FASN 0.059 0.606 0.251 0.036 ACC 0.062 0.591 0.240 0.042

Human and rodent obesity associated with reduced pRb activity in adipose tissue. Weight loss restored adipose pRb activity in rats

BMI <30

BMI >30

←pThr821 RB1

← total RB1

Normal fa

t

High fat

Cafete

ria

Cafete

ria re

versi

on0

50

100

150

200 *#

pSer780 Rb1 →

total Rb1 →

p=0.02

phospho RB1/ total RB1 in SAT (similar result for VAT, 95% CI)

BMI <30 BMI >30

phospho Rb1/ total Rb (% NF)

Human cohort Animal cohort








adipose tissue?


mice increased?



metabolism



Specific objectives

CENIT–PRONAOS

DIABATBIOCLAIMS

CIBERobn

Glucose and fat tolerance test, insulin and leptin sensitivity test, indirect calorimetry

Circulating parameters determination

Gene expression analysis in white, brown adipose tissue and in muscle

Body composition analysis

Age, months

- - - - -

----- -----

1 2 3 4 5 6 7

Body weight and energy intake - continuously monitored.

Rb+/- and WT male mice fed a standard semi-synthetic diet (10% of calories from fat) were studied from weaning until the age of 6-7 months.

Experimental design

Rb+/- mice displayed reduced adiposity at mature adult age and improved blood lipids

0 45 90 135 180 22565

70

75

80

85lean body mass

age (days)(g

/100

g B

W)

*

0 45 90 135 180 22515

20

25

30

35body weight

age (days)

(g)

0 45 90 135 180 2255

10

15

20

25

body fat massWT

age (days)

(g/1

00 g

BW

)

*

young adult0.0

0.4

0.8

1.2

1.6fasting NEFA

(mM

)

*

* *

young adult0.0

0.1

0.2

0.3

0.4

fasting TAGs

(mM

)

*

* *

* P<0.05 different from WT

liver of adult mice

WT Rb+/-

Age-associated increases in fasting plasma insulin, glucose levels and HOMA-IR index were attenuated in Rb+/- mice

young adult0369

12

fasting glucoseWTRb+/-

(mM

)*

#

young adult0

50100150200250

fasting insulinWTRb+/-

(pM

)

*

#*

young adult0.0

0.1

0.2

0.3

0.4

0.5

R-QUICKI WTRb+/-

(a.u

.)

*

# #

young adult0

4

8

12

16

HOMA-IR WTRb+/-

(a.u

.)

*

#*

* P<0.05 different from WT; # P<0.05 different from young age, t-test

0 30 60 90 120 150 18005

101520253035

time (min)

gluc

ose

(mM

)

0 30 60 90 120 150 18005

101520253035

time (min)

gluc

ose

(mM

)

* *

Rb+/- mice displayed increased sensitivity to exogenous insulin (in ITT) and to glucose (in GTT)

0 15 30 45 60 75 900

20

40

60

80

100

120

time (min)

gluc

ose

(%)

* *

0 15 30 45 60 75 900

20

40

60

80

100

120

WT Rb+/-

time (min)

gluc

ose

(%)

* * * *

* P<0.05 different from WT

young

adult

sensitivity to insulin tolerance to glucose

0

25

50

75

100

AUC

(x10

0)*

*

0

25

50

75

100

AUC

(x10

0)

*

*

0

25

50

75

100

AUC

(x10

00)

*

0

25

50

75

100

AUC

(x10

00)

*

##

* P<0.05 different from WT, # P<0.05 different from young age

WT Rb+/-0

25

50

75

100

125

150young saline

leptin

24h

cum

ulati

vefo

od in

take

(g/k

g BW

)*

‡

WT Rb+/-0

25

50

75

100

125

150 adultsalineleptin

12h

cum

ulati

vefo

od in

take

(g/k

g BW

)

*

‡

0 60 120 180 2400.0

1.0

2.0

3.0

4.0young WT

time (min)

tria

cylg

lyce

rols

(mM

)

0 60 120 180 2400.0

0.5

1.0

1.5

2.0adult WT

time (min)

tria

cylg

lyce

rols

(mM

)

*

leptin sensitivity test oral fat tolerance test

‡different from saline, P <0.05, t-test *different from WT, P <0.05, t-test

0.75

0.85

0.95

1.05adult

time

RER

dark** ***** * ****

0.75

0.85

0.95

1.05young

WT Rb+/-

time

RER

dark*** * * **

***

* different from WT, P <0.05, t-test.

Preferential use of fatty acids as a fuel and increased metabolism in Rb+/- mice

indirect calorimetry

young36

37

38

39

40

(°C)

*

*

adult

*

*

Series132

33

34

35

36

(°C)

*

after 6h fast

after 3h cold exposure

rectal temperatureWTRb+/‒

250

750

*

0

80

160

adult WTRb+/-

*

** *

500

1000

**

0100200300

young WTRb+/-

*

0100200300

young WTRb+/-

*

**

*

400

800

*

0

100

200

300

400adult WT Rb+/-

*

**

*

retroperitoneal WAT epididymal WAT

WAT depots of Rb+/- mice displayed an increased capacity for fatty acid oxidation and thermogenesis

*different from WT, P <0.05, t-test

youngWT Rb+/- WT Rb+/-

adult

1.0±0.1 0.6±0.2 1.0±0.6 14±4.3*

1.0±0.05 0.8±0.03 1.0±0.03 1.4±0.1*

1.0±0.04 1.4±0.2 1.0±0.1 2.1±0.5

UCP1 Ctr

PPARα Ctr

CPT1-b amido black

Rb +/-

anti-UCP1

WT anti-UCP1

Rb +/- anti-UCP1

iWAT

imm

unoh

istoc

hem

istry

imm

unob

lotti

ng

*different from WT, P <0.05, t-test.

500

1000

*

*

Pgc-1α

Cpt1b Cidea Ucp10

50

100

150

young WT Rb+/-

mRN

A (%

)

*

**

Pgc-1α Cpt1b Cidea Ucp10

255075

100125

adult

mRN

A (%

)

*

PGC-1 Ctr


adult

UCP1 Ctr

1.0±0.03 1.3±0.1* 1.0±0.1 0.8±0.02

1.0±0.1 0.9±0.1 1.0±0.1 0.8±0.1

Young, but not adult, Rb+/- mice had increased capacity for fatty acid oxidation/thermogenesis in BAT

mRNA expression protein expression

*different from WT, P <0.05, t-test.

Ppard Pgc-1α Cpt1b Pdk4 Ucp30

50

100

150

200

250young

WT Rb+/-

mRN

A (%

)

*

Ppard Pgc-1α Cpt1b Pdk4 Ucp30

4080

120160200 adult

mRN

A (%

)

*

* *

UCP3 Ctr


adult

CPT1β amido black

1.0±0.2 1.8±0.1*

1.0±0.2 1.5±0.2

1.0±0.3 2.3±0.3*

1.0±0.4 2.1±0.7

Increased capacity for fatty acid oxidation in skeletal muscle of Rb+/-mice

mRNA expression protein expression








metabolism?


adipose tissue?


mice increased?



metabolism



Specific objectives

CENIT–PRONAOS

DIABATBIOCLAIMS

CIBERobn

insulin15-30min

basal and insulin-

stimulated glucose uptake

flux analysis (SeaHorse)

lipid accumulation

(Oil Red)


detach

C2C12 myofibroblasts

reverse-transfect

with siRNA

protein and total RNA isolation; qPCR; immunoblotting; immunofluorescence

replate for 28-36h

myotubesCtrRB1

siNON siRb

silenced myotubes

Insulin6h

Experimental design

0 4 8 12 16 200

time (h)0 25 50 75 100

50100150200250300

palmitate

**

* * * * * *

80

100

120

140 oleate *

Cd36 Cpt1b Pdk4 Acc Mcad Acox1 PpardPpargc1a Ucp3 Lpl Lipe Pnpla20

100

200

mRN

A (%

) **

* ** *

**

*

siNONsiRb

Rb1 silencing in C2C12 myotubes increased fatty acid catabolism

time (min)

siNONsiRb

oxygen consumption rate (%) lipid accumulation(%)

basal mRNA expression


Rb silencing in C2C12 myotubes enhanced mitochondria

mtDNA/gDNA (%)

mitofusin 2 protein

expression (%)

*, P < 0.05, siRb versus siNON; Student’s t test

siNONsiRbWim Mandemakers

et al. 2007

0255075

100125150175

*

*

0

50

100

150

200

250

300

*

*

Glut40

200

400

600

mRN

A (%

)

* siNONsiRb

Rb silencing in C2C12 myotubes increased glucose uptake in a Glut4-dependent manner

siNONsiRbsiNON + insulinsiRb + insulin

*, P<0.05, siRb versus siNON; #, P < 0.05, insulin versus vehicle; †, P < 0.05, phloretin versus vehicle, t test

- phloretin + phloretin0

10

20

30

2-DG

upt

ake

(pm

ol/m

in/w

ell)

*#

*

basal and insulin-stimulated protein expression of Glut4

basal and insulin - stimulated glucose uptake

Glut1 Hk2 Pdh1a

* *


+ insulin

siNON

siRb

Rb p107 p130 Pdk4 Lpl Lipe Pnpla2 Glut10

50100150200250 * #

#

* *

*

# #*#

#

* *#

#

*

# #

# #**##*

mRNA expression of pocket proteins and genes transcriptionally controlled by insulin (6h)

siNON

mRN

A (%

)

Series10

100

200

300

400 * ##

Rb1 silencing in C2C12 myotubes did not compromise insulin sensitivity

pAKT/AKT (%)

basal and insulin (30min)-dependent AKT phosphorylation

*, P < 0.05, siRb versus siNON; #, P < 0.05, insulin versus vehicle, Student’s t test


mRN

A (%

)

siNON + insulinsiRb siRb + insulinInsr Irs1 Pi3k Socs3

020406080

100120

rest exercisep-pRb (Ser780) p-pRb(Ser780) p-pRb (Ser780)

gastrocnemius muscle testis (control)

Exercise in mice led to inactivation of pRb in skeletal muscle

WT mice subjected to acute exercise on a treadmill

1 2 3 4 5 60

255075

100

posi

tive

nucl

ei

/ nuc

lei (

%)

restexercise

*

rest exercise

p-pRb (Ser780) positive nuclei / nuclei (%)








adipose tissue?





metabolism



Specific objectives

CENIT–PRONAOS

DIABATBIOCLAIMS

CIBERobn

WT Rb+/-

BAT

gWAT iWAT

enrichment for SVF

fow cytometry of Sca1+ labelled crude suspension


2 months old

differentiated primary

adipocytes

iWAT SVF

WT

crude suspension

SVF

total RNA isolation; qPCR

label with FITC anti-Sca1

enrichment for Sca1+

Sca1+ preadipocytes

10%

13%

+/- Rosi

Experimental design

Rb+/-

10007000 # # # # ##

##

## #

20000350005000065000

#

PpargFa

bp4Le

pUcp

1

Prdm16

Ppargc1a

Ppargc1b

PparaCpt1b

Cd137

Tmem26Tbx1

Slc27a1

Hoxc9

Shox2

Slc2a1

Slc2a4

0300600900

*#

#

#

#

##

##

#

##

#

PpargFa

bp4Le

pUcp

1

Prdm16

Ppargc1a

Ppargc1b

PparaCpt1b

Cd137

Tmem26Tbx1

Slc27a1

Hoxc9

Shox2

Slc2a1

Slc2a4

0

100

200

300

**

*

***

*

WAT primary cultures from Rb+/- mice displayed upregulation of thermogenesis-related genes but not of beige-specific markers

adipocyte markers

brown adipocyte function/thermogenesis

beige adipocyte markers

GLUTs

WTRb+/‒

(basal conditions)

(+ rosiglitazone)

adipocytes from iWAT SVF

WAT primary cultures from Rb+/- mice display increased basal glucose uptake

+ rosiglitazonebasal conditionsSeries1

0

50

100

150

200

* ††

Se-ries1

0

50

100

150

200

2-DG

upt

ake

(pm

ol/h

/µg

prot

.)

*† WT

Rb+/‒

WT + insulinRb+/‒ + insulin

basal and insulin - stimulated glucose uptake

D. (gWAT)

PpargCebpa

Zeb1

Prdm16

Ppargc1

a

Ppargc1

bCebpb

Ebf2

Foxc2

Tbx1

Rip140

Twist

-1

Tmem26

Cd137Pdgfr

a

Bmpr1a

Bmpr1b

Bmpr2Acvr

10

50

100

150

200

**

** **

*

adipogenesis brown and/or beige adipogenic program

surface markers

receptors for BMPs

* different from WT, P<0.05, Student’s t test

Sca1+ preadipocytes from Rb+/- mice have increased expression of transcriptional modulators of

brown/beige adipogenesis and of BMPs receptors

mRN

A (%

)

iWAT

Series1

*

2m-old

BMP7 in serum (pg/mL)

7m-oldSeries1

0

100

200

* different from WT, P<0.05, Student’s t test

Shox2 Cd137 Tmem26 Slc27a1 Tbx1 Hoxc9 Fgf21 Pdgfra0

50

100

150m

RNA

(%)

* ** *

*

WTRb+/‒rWAT of 7m-old mice

Reduced expression of beige- specific markers in WAT of Rb+/- mice

Increased circulating BMP7 in Rb+/- mice








adipose tissue?


mice increased?



metabolism



Specific objectives

CENIT–PRONAOS

DIABATBIOCLAIMS

CIBERobn

Mouse embryonic fibroblasts

(MEFs)

non-adipogenic mediaDo treatments

affect the differentiation fate

of MEFs? (adipogenic/ chondrogenic

balance)

adipogenic induction

vehicle hyaluronic acid (HA)dermatan sulfate (DS)HA+DS mix

Experimental design Control

GAGs

Do treatments affect adipogenesis?

pRb status?

Prdm16 Ppargc1a Ppargc1b Ppara Nrf1 Cpt1b Ucp1 mt-Co2 Cox5a0

50100150200250

mRN

A (%

) ****

020406080

100120

* * * *

PPARγ C/EBPα FASN

vehicle HA

DS HA+DS *Se-

ries1total Rb pRb/Rb

*

*Oil Red

vehicle HA 160 mg/mL DS 40 mg/mL HA80+DS20 200 mg/mL

Down-regulation of pRb associated with reduced adipogenesis but not increased browning in GAG-treated MEFs

protein (%)

* different from vehicle, P<0.05, Student’s t test








adipose tissue?


mice increased?



metabolism



Specific objectives

CENIT–PRONAOS

DIABATBIOCLAIMS

CIBERobn

early biomarker of health

Identification of early biomarkers of health

health

time

transcriptome analysis in models of healthier ageing vs control

challenge tests

controlmodels of metabolic robustness and healthy ageing

classical biomarkerpredicts the incidence of outcome or disease

blood?

Rb+/- model?

months

WT and Rb+/-

male mice, fed on normal fat,

maintenance diet

total blood extraction, body composition

0 1 2 3 4 5 6 7



Prevalidation study

Does whole blood can reflect transcriptional response to fasting as do PBMC in rats?

Do changes in gene expression in total blood of ATRA-treated mice reproduce known ATRA effects on gene expression in liver and brown adipose tissue?

Yes!

Yes!

store total blood in RNALater until all samples are obtained

total RNA isolation and qPCR

Experimental design

Fasn Lrp1 Rxrb Sorl10

50

100

150WT Rb

mRN

A (%

)

* * * *

+/-


50100150

mRN

A (%

)


50

100

150

mRN

A (%

)

Putative transcript-based biomarkers of metabolic robustness were verified in total blood of young Rb+/-mice

total blood gene expression

Whole blood RNA as a source of transcript-based nutrition and metabolic health-related biomarkers

Petar D. Petrov, M. Luisa Bonet, Barbara Reynés, Paula Oliver, Andreu Palou, Joan Ribot

Submitted manuscript







metabolism?


adipose tissue?





metabolism



Specific objectives

CENIT–PRONAOS

DIABAT

BIOCLAIMS

CIBERobn

All in all, work in this thesis reveals a translational potential of pRb modulation for improvement of

metabolic health.

Identification of physiological factors and of interventions capable of maintaining pRb

expression/activity to an "optimal" level in fat, muscle and other relevant tissues could contribute to a

healthier phenotype.

Conclusiones



I. La proteína del retinoblastoma está involucrada en la adipogénesis humana.

II. La proteína del retinoblastoma está involucrada en el mantenimiento del fenotipo del adipocito diferenciado.

III. La obesidad humana y murina afecta el estatus de la proteína del retinoblastoma en tejidos adiposos blancos.

V. La haploinsuficiencia del gen de la proteína del retinoblastoma confiere ventajas metabólicas frente a diferentes formas de estrés fisiológico agudo y reduce la acumulación de grasa corporal y el deterioro metabólico que habitualmente acompañan el tránsito entre la edad joven y la de adulto maduro.

IV. La expresión y actividad de la proteína del retinoblastoma en adipocitos se ve modulada por compuestos con actividad anti-adipogénica; en cambio, la reducción de la expresión/actividad de la pRb no se puede emplear como un marcador inequívoco del proceso de “marronizacion”.

VI. Los preadipocitos residentes en el tejido adiposo blanco de los ratones Rb+/- retienen una capacidad incrementada para la adipogénesis marrón, pero no para la adipogénesis beige, y presentan una capacidad incrementada de respuesta a proteínas morfogenéticas del hueso (BMPs).

VIII. Niveles reducidos en sangre de los ARNm de Fasn, Lrp1, Rxrb y Sorl1 han sido verificados como posibles biomarcadores predictivos de salud metabólica mejorada, utilizando el modelo murino de haploinsuficiencia del gen de la proteína del retinoblastoma.

VII. El estatus de la proteína de retinoblastoma en el musculo esquelético puede jugar un papel en el control del metabolismo muscular y en la adaptación del mismo al ejercicio, mediante efectos que la pRb ejerce sobre la utilización de sustratos en este tejido.



Petar Petrov 5 February 2016, Palma de Mallorca, Spain

65

Supplementary data (not presented during the

doctoral defense)

Glut4

Glucose

CD36Fatty acids

PDH is a key metabolic switch between glucose and FA utilization and pRB may be indirectly involved in its control by

affecting its suppressor PDK

pRb

Hsieh et al., 2008

E2F1

pRb status and expression can be modulated by bioactive compounds

еllagic acid

all-trans retinoic acid

Bioactives inhibiting cell proliferation

Bioactives inhibiting

adipogenesis or promoting WAT

browning

curcuminsilibininlunasin

lactoferrin berberine

pRb

68

GAGs mix inhibits spontaneous adipogenesis in MEFs and induces chondrocyte- related gene expression in MEFs

vehicle HA

DS HA+DS Pparg Cebpa Fasn Lep

0

20

40

60

80

100

120

mRN

A (%

)

** *

n.d.

010203040506070

*

*

(a.u

.)

vehicle HA 80 g/mLDS 20 g/mL HA80+DS20 100 g/mL

BMP2 100 ng/mL

DS BMP2vehicle HA HA+DS

Acan mRNA

Immunofluorescence for Acan

Spontaneous adipogenesis

69

siNONsiRb

Rb p107 p1300

50

100

150

200

250m

RNA

(%)

*

*

RBp107Ctr

siNON siRb

100 kDa –

45 kDa –

100 kDa –

siRNA based reverse-silencing of pRb efficiently reduced mRNA and protein levels of pRb without affecting the myotubes´ morphology

Day 1 siNON siRbDay 5Day 7

44h post reverse-transfection

just before reverse-

transfection

70

Insr Irs1Pik3r1

Socs3 Glut40

50100150200

WT 2 mRb 2 mWT 7 mRb 7 mm

RNA

(%)

* *

muscle

The enhanced insulin sensitivity of Rb+/- mice was partially reflected by changes in mRNA expression of genes involved in the insulin signalling pathway.

*

Insr Irs1 Pik3r1 Socs3 Glut40

50

100

150

mRN

A (%

) *eWAT

Insr Irs1 Pik3r1 Socs3 Glut40

100

200

300

mRN

A(%

)

* *

rWAT

*

*Different from WT, P 0.05, t-test

Supplementary data

PGC1 alpha FASN PPAR alpha Srebp1C0

20

40

60

80

100

120

140

160 Gene expression in mouse male liverMean WT 2 monthsMean Rb+/- 2 monthsMean WT 6 monthsMean Rb+/- 6 months

Supplementary data

72

Regulation of Glut4 promoter is complex and we proposed amechanism to explain the observed induction of GLUT4

TRE

-408 -388

T3Ra

Trip230Rb1

enhances

inhibits

Silencing of Rb

G0

250

500

mRN

A (%

)

*

*

siNONsiRb

TRE

-408 -388

TRE

-408 -388

T3Ra

Trip230

Rb1

enhances

inhibits

thyroid hormonereceptor

hyroid hormone receptor coactivator

73

Complex formation

pRb has a dual role in white (murine) adipogenesis

Mesenchymal precursor

Committed preadipocyte

Growth-arrested preadipocyte

Mitotic clonal expansion

Terminal differentiation

Mature adipocyte

Stimuli (IDM)

↑C/EBPβ , C/EBPδ

↑ C/EBPα, ↑PPARγ

pRb

pRb

pRb phosphorylation

(deactivation)

pRbpRb

diffe

renti

ation

Dediferentiated adipocyteObesity TNFα

pRb ???

↑ SREBP1c

↑ PPARα

Rb1 #190

p107

newp130

AdipoqLep

tin

newCd36

Pgc1a

Pgc1b

Ap2Cpt1b

Fasn Lp

lPparg

Tmem

26Hoxc9

Ppara Ucp1

Glut1 Glut4

0

50

100

150

200

250

Silencing of pRb1, p107 and p130 in differentiated (Day 7) 3T3-L1 adipocytes

siNON siRb1 sip107 sip130

*

*

*

*

*

*

~

*

**

*

~

*

*

75

E2F

pRB

Oxidative metabolism

BAT, WAT, SM

Kir 6.2

Hyperglycaemia

Insulin

The involvement of pRb in metabolic control can in part be due to its ability to affect the activity of E2Fs. pRB and E2F1 are connected to insulin

Blanchet et al., 2011 Hansen et al., 2004,Dali-Youcef et al. ,2007

Annicotte et al., 2009Insulin

pancreas

pRBCDK4

E2F

Doctoral thesis defense Petar Petrov version 5 Feb 2016

Documents

Transcript of Doctoral thesis defense Petar Petrov version 5 Feb 2016