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
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
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
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
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
youngWT Rb+/- WT Rb+/-
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
youngWT Rb+/- WT Rb+/-
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
*different from WT, P <0.05, t-test
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
DIABATBIOCLAIMS
CIBERobn
insulin15-30min
basal and insulin-
stimulated glucose uptake
flux analysis (SeaHorse)
lipid accumulation
(Oil Red)
differentiate for 5 days
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
*different from WT, P <0.05, t-test
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
* *
basal mRNA expression
+ 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
basal mRNA expression
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 (%)
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
DIABATBIOCLAIMS
CIBERobn
WT Rb+/-
BAT
gWAT iWAT
enrichment for SVF
fow cytometry of Sca1+ labelled crude suspension
differentiate for 7 days
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
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
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
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
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
total blood extraction, body composition
total blood extraction, body composition
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 (%
)
* * * *
+/-
Fasn Lrp1 Rxrb Sorl10
50100150
mRN
A (%
)
Fasn Lrp1 Rxrb Sorl10
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
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
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
CHARACTERIZATION OF THE ROLE OFRETINOBLASTOMA PROTEIN IN METABOLIC REGULATION
Doctoral Thesis Defense
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.
CHARACTERIZATION OF THE ROLE OFRETINOBLASTOMA PROTEIN IN METABOLIC REGULATION
Doctoral Thesis Defense
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