Brown adipose tissue:
function and physiological significance
Barbara Cannon
The Wenner-Gren Institute, Stockholm University
Results in collaboration with
(among others)
Wenner-Gren Institute
Stockholm University
Tore Bengtsson
Valeria Golozoubova
Helena Feldmann
Tomas Waldén
Natasa Petrovic
Irina G. Shabalina
and
Jan Nedergaard
Royal Veterinary College
James Timmons
Karolinska Institute
Ola Larsson
University of Ancona
Saverio Cinti
Maria Cristina Zingaretti
Mice in the cold
Body temperature
40
60
Food consumption
Metabolism
8
6
30
40
g/mouse
ml O2/(min•kg0.75)
T b, °C
35
20
RMR
25
0
60
120
Time at 4°, min
180
2
0
0
20
4
0
10
20
30
Temperature, °C
0
10
20
30
Temperature, °C
40
Acute cold exposure of control animals
Shivering during
acute Shivering
cold exposure
Heat production
60
18°-acclimated at 4°
150
100
µV
at 4°
50
at 30°
ml O2/(min•kg0.75)
WT
at 4
40
20
at 30
RMR(WT)
0
0
1 min
20 min
with Eso Hohtola
The paradigm of classical* nonshivering thermogenesis:
with time in cold (weeks),
shivering ceases
"Shivering" after
acclimation to cold
Shivering during
acute cold exposure
150
100
100
µV
at 4°
50
µV
150
50
at 4°
at 30°
at 30°
0
1 min
0
1 min
*cold-acclimation recruited, cold-induced
but metabolism remains high:
acutely in cold
60
18°-acclimated at 4°
60
Cold
wks at 4
afteracclimated
3 weeks (>3
in cold
WT
ml O2/(min•kg0.75)
ml O2/(min•kg0.75)
WT
40
20
20
at 30
RMR(WT)
RMR(WT)
0
at 4
40
0
20 min
20 min
Enerbäck//Kozak 1997
Wild-type mice
"Shivering" after
acclimation to cold
Shivering during
acute cold exposure
150
150
100
µV
µV
100
at 4°
50
50
at 4°
at 30°
at 30°
0
0
1 min
1 min
UCP1(-/-) mice
150
100
100
µV
at 4°
at 4°
µV
150
50
50
at 30°
0
1 min
at 30°
0
1 min
Wild-type mice
"Shivering" after
acclimation to cold
Shivering during
acute cold exposure
150
150
100
µV
µV
100
at 4°
50
50
at 4°
at 30°
at 30°
0
0
1 min
1 min
UCP1(-/-) mice
150
100
100
µV
at 4°
at 4°
µV
150
50
50
at 30°
0
1 min
at 30°
0
1 min
And metabolism remains high
60
in the
B. Acutely
18°-acclimated
at cold
4°
60
C. Cold
Cold
acclimated
40
UCP1(-/-)
20
RMR(UCP(-/-))
40
UCP(-/-)
20
RMR(UCP(-/-))
RMR(WT)
RMR(WT)
0
WT
ml O2/(min•kg0.75)
ml O2/(min•kg0.75)
WT
0
20 min
20 min
Thus, the presence or absence of brown fat
does not alter the amount of extra energy spent in the cold
(but it is more comfortable to spend the energy
if you have brown fat)
Wild-type mice
"Shivering" after
acclimation to cold
Shivering during
acute cold exposure
150
150
100
µV
µV
100
at 4°
50
50
at 4°
at 30°
at 30°
0
0
1 min
1 min
UCP1(-/-) mice
150
150
at 4°
µV
at 4°
µV
100
100
50
50
at 30°
0
No cold-induced
nonshivering
thermogenesis
without UCP1
1 min
at 30°
0
1 min
Does the presence of UCP1
affect
”adaptive adrenergic
nonshivering thermogenesis”?
NE injection
NE
30°C
Adapted to 30 °C
Adapted to 4 °C
50
50
30 °C
Oxygen
consumption
(thermogenesis)
wildtype
40
NE
NE
NE
20
wi ldtype
m l O2 • min • kg0.75
m l O2 • min • kg0.75
40
30
4 °C
30
NE
20
UCP1 KO
10
0
UCP1 KO
10
0
Adapted to 30 °C
Adapted to 4 °C
50
50
30 °C
Oxygen
consumption
(thermogenesis)
wildtype
40
NE
NE
NE
20
wi ldtype
m l O2 • min • kg0.75
m l O2 • min • kg0.75
40
30
4 °C
30
NE
20
UCP1 KO
10
0
UCP1 KO
10
0
Adapted to 30 °C
Adapted to 4 °C
50
50
30 °C
Oxygen
consumption
(thermogenesis)
wildtype
40
NE
NE
NE
20
wi ldtype
m l O2 • min • kg0.75
m l O2 • min • kg0.75
40
30
4 °C
30
NE
20
UCP1 KO
10
0
UCP1 KO
10
0
Adapted to 30 °C
Adapted to 4 °C
50
50
30 °C
Oxygen
consumption
(thermogenesis)
wildtype
40
NE
NE
NE
20
wi ldtype
m l O2 • min • kg0.75
m l O2 • min • kg0.75
40
30
4 °C
30
NE
20
UCP1 KO
10
0
UCP1 KO
10
0
Adapted to 30 °C
Adapted to 4 °C
50
50
30 °C
Oxygen
consumption
(thermogenesis)
wildtype
40
NE
NE
NE
20
wi ldtype
m l O2 • min • kg0.75
m l O2 • min • kg0.75
40
30
4 °C
30
NE
20
UCP1 KO
10
0
UCP1 KO
10
0
Adapted to 30 °C
Adapted to 4 °C
50
50
30 °C
Oxygen
consumption
(thermogenesis)
wildtype
40
NE
NE
NE
20
wi ldtype
m l O2 • min • kg0.75
m l O2 • min • kg0.75
40
30
4 °C
30
NE
20
UCP1 KO
10
0
UCP1 KO
10
0
No cold-recruited adaptive adrenergic
nonshivering thermogenesis without UCP1
The uncoupling protein(-1), UCP1
As determined from gene synteny, it would seem that
UCP1 developed from a protoUCP1, found e.g.
in fish. ProtoUCP1 has no thermogenic function.
The evolution was thus dramatic.
carrier
dicarboxylate
ornithine
carrier BMCPs
(UCP5)
carnitine
carrier
UCP4
M
i
t
o
c
h
ophosphate
n carrier
d
r
i
a
l
oxoglutarate
carrier
UCP1
UCP2
birdUCPs
UCP3
plantUCPs
Graves'
disease
antigen
citrate
carrier
sulfate
carrier
ATP/ADP
carriers
(Borecky, Maia, Arruda (2001))
conserved
in all
mitochondrial
carriers
conserved
in
all
UCPs
conserved
in all
mitochondrial
carriers
conserved in all UCP1s only
conserved
in
all
UCPs
conserved
in all
mitochondrial
carriers
conserved in all UCP1s only
The unique sequences
of UCP1
may correlate
with unique function:
mediation of adaptive
thermogenesis
UCP1 is essential
for norepinephrine-induced thermogenesis
in brown adipocytes
Oxygen consumption rate
fmol O 2 / (min • cell)
500
400
300
Brown adipocytes
with UCP1:
high thermogenic capacity
NE
200
100
0
No effect on basal:
no inherent leakiness
of UCP1
No UCP1:
no thermogenesis
1 min
FFA
B. Fatty-acid-induced
thermogenesis
500
Brown adipocytes
from normal animals
Oxygen consumption rate
400
300
oleate
200
100
0
Brown adipocytes
from animals
without UCP1
1 min
FFA
B. Fatty-acid-induced
thermogenesis
B. Fatty-acid-induced
thermogenesis
500
500
Brown adipocytes
from normal animals
300
oleate
200
100
0
Brown adipocytes
from animals
without UCP1
1 min
400
Oxygen consumption rate
Oxygen consumption rate
400
Brown adipocytes
from normal animals
300
oleate
200
100
0
Brown adipocytes
from animals
without UCP1
1 min
Bartelt//Heeren 2011
Brown-fat cells:
Thermogenesis
log[NE]
Glucose uptake
log[NE]
Marette & Bukowiecki 1991
Bartelt//Heeren 2011
Bartelt//Heeren 2011
Brown and white fat cells:
alike or different?
John Horowitz
White
Brown
Brown and white fat cells:
alike or different?
John Horowitz
Are the cells different due to external ”forces”
- or are they inherently different?
Cell culture:
brown versus white
precursors from
brown adipose tissue
white adipose tissue
(interscapular)
(epidydimal)
identical
culture
conditions
differentiate in culture
examine global gene expression
White precursors
4
6
Brown precursors
8
10 days in culture
Rat cells
Mature brown and white adipocytes
express different genes (!)
white
(3 different
cell cultures)
brown
(2 different
cell cultures)
brown and white
remain different
under identical
Even before differentiation
brown and white adipocytes
express different genes
white
(3 different
cell cultures
brown
(3 different
cell cultures)
Genes enriched >5 fold
in brown versus white undifferentiated pre-adipocytes
Gene Symbol
Gene Title
Acta1
actin, alpha 1, skeletal muscle
Actc1
actin, alpha, cardiac
Cd83
CD83 antigen
Chrna1
cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle)
Cldn5
claudin 5
Icam2
intercellular adhesion molecule 2
Lhx8
LIM homeobox protein 8
Meox2
mesenchyme homeobox 2
Mme
membrane metallo endopeptidase
Myh3
myosin, heavy polypeptide 3, skeletal muscle, embryonic
Myl1
myosin, light polypeptide 1
Mylpf
myosin light chain, phosphorylatable, fast skeletal muscle
Myog
myogenin
Tbx15
T-box 15
Tnnc1
troponin C, cardiac/slow skeletal
Tnni1
troponin I, skeletal, slow 1
Tnnt3
troponin T3, skeletal, fast
Zic1
zinc finger protein of the cerebellum 1
Myogenin
Myf5
MyoD
Myf6
A myogenic gene expression signature establishes
that brown and white adipocytes originate from distinct cell lineages
A myogenic gene expression signature establishes
that brown and white adipocytes originate from distinct cell lineages
Timmons et al. (2007) PNAS
muscle
Myogenin
Myf5
MyoD
Myf6
Myf5 is expressed in both muscle and brown-fat cells
(but disappears during maturation of brown-fat cells).
Myogenin
A cell that
has at one time expressed Myf5 MyoD
is therefore marked as coming from the muscle/brown-fat line
Myf5
Myf6
Nature 2008
Thus, even in the intact mouse,
the brown-fat cells
(and of course the muscle cells)
have at some time expressed myf5
and brown-fat and muscle
thus originate
from the same lineage
A myogenic gene expression signature establishes
that brown and white adipocytes originate from distinct cell lineages
Timmons et al. (2007) PNAS
muscle
mesenchymal
stem cell
adipomyocyte
and myocyte
common precursor
Myf5
PRDM16
miR-206
UCP1
”adipomyocyte”
classical brown adipocyte
miR-206
myocyte
mesenchymal
stem cell
adipomyocyte
and myocyte
common precursor
white preadipocyte and
brite adipocyte
common precursor
Myf5
Hoxc9
Hoxc9
Tcf21
white adipocyte
Hoxc9
UCP1
”brite” adipocyte
PRDM16
miR-206
UCP1
miR-206
myocyte
”adipomyocyte”
classical brown adipocyte
Petrovic et al JBC 2010
Before 2007:
” in man, brown adipose tissue (UCP1)
is only found in newborns”
2007:
Symmetrical tumours?
Tense muscle?
Fatty tissue - CT scans
www.med.harvard.edu/
JPNM/chetan/normals
After 2007:
Established that
adult humans (may) have brown fat
but
- how many (what is the prevalence)?
- how much do they have?
- does it matter?
Feels cold:
Christensen et al. 2006
Feels cold:
Feels warm
Christensen et al. 2006
The tissue really is brown fat as it contains UCP1
It is found in 1/3 of all patients
Zingaretti et al. 2009
highly sympathetically innervated
C. Sympathetic nerve density
0,015
Proportion of field
0,010
0,005
0,000
Brown islands
White areas
Zingaretti et al. 2009
Typical mitochondria
Zingaretti et al. 2009
Even precursor cells….
Thus adult humans possess
UCP1-containing brown fat!
Does it matter
whether we have brown fat or not?
Functional evidence only from mice
Wildtype mice
UCP1 KO mice
Would the presence/absence
of UCP1/brown fat affect
basal metabolism?
UCP1 is essential
for norepinephrine-induced thermogenesis
in brown adipocytes
Oxygen consumption rate
fmol O 2 / (min • cell)
500
400
300
Brown adipocytes
with UCP1:
high thermogenic capacity
NE
200
100
0
No effect on basal:
no inherent leakiness
of UCP1
No UCP1:
no thermogenesis
1 min
Indirect calorimetry
Thus, in mice, basal metabolism is not affected
by the amount of brown fat
- and would not be affected in humans
(Thus only to get more brown fat doesn’t ”help”
- it must also be activated)
Would the presence/absence
of brown fat affect
”diet-induced thermogenesis”
Would the presence/absence
of brown fat affect
diet-adaptation-recruited
norepinephrine-induced
thermogenesis?
No diet-recruited adaptive adrenergic
nonshivering thermogenesis without
brown fat
No diet-recruited adaptive adrenergic
nonshivering thermogenesis without
brown fat
+
No cold-recruited adaptive adrenergic
nonshivering thermogenesis without brown fat
=
No adaptive adrenergic
nonshivering thermogenesis without brown fat
What are the consequences
of lack of
diet-adaptation-recruited
norepinephrine-induced
thermogenesis
exercise
muscle tension?
shivering
WT
WT
KO
WT
KO
WT
Without
brown fat
mice
become
fatter
Without
brown fat
mice
become
fatter
at thermoneutrality!
So - is this relevant
for human metabolism?
A. Presence of BAT versus BMI
8
7
Numbers
6
5
4
3
2
1
0
15
20
25
30
35
40
BMI
Zingaretti et al., 2009
Present in the younger and slimmer (!)
B. Presence of BAT versus age
8
8
7
7
6
6
5
5
Numbers
Numbers
A. Presence of BAT versus BMI
4
3
4
3
2
2
1
1
0
0
15
20
25
30
BMI
35
40
20
30
40
50
60
70
80
90
Age
Zingaretti et al., 2009
Present in the younger and slimmer (!)
B. Presence of BAT versus age
8
7
7
6
6
5
5
Numbers
8
4
3
4
3
2
2
1
1
0
0
15
20
25
30
35
40
20
30
40
BMI
50
60
70
80
90
Age
C. BAT versus BMI and age
40
35
30
BMI
Numbers
A. Presence of BAT versus BMI
25
20
15
10
20
30
40
50
Age
60
70
80
90
Zingaretti et al., 2009
Present in the younger and slimmer (!)
C. BAT versus BMI and age
40
35
BMI
30
25
20
15
10
20
30
40
50
60
70
80
90
Age
Zingaretti et al., 2009
≈ 100
fold
difference
Saito et al. 2009
Evidence from man
The -3826
polymorphism
determines
UCP1 expression
Evidence from man
Correlation of UCP1 genotype with obesity
Conclusions
decreased adaptive thermogenesis
– can be causative of obesity
adaptive thermogenesis
– does counteract obesity
activation of adaptive thermogenesis
– may become an antiobesity tool
– perhaps even an anti-diabetes tool
but is there anything already here
that really shows that the cell types
are different?
UCP1
Genes enriched >5 fold
in brown versus white undifferentiated pre-adipocytes
Gene Symbol
Gene Title
Acta1
actin, alpha 1, skeletal muscle
Actc1
actin, alpha, cardiac
Cd83
CD83 antigen
Chrna1
cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle)
Cldn5
claudin 5
Icam2
intercellular adhesion molecule 2
Lhx8
LIM homeobox protein 8
Meox2
mesenchyme homeobox 2
Mme
membrane metallo endopeptidase
Myh3
myosin, heavy polypeptide 3, skeletal muscle, embryonic
Myl1
myosin, light polypeptide 1
Mylpf
myosin light chain, phosphorylatable, fast skeletal muscle
Myog
myogenin
Tbx15
T-box 15
Tnnc1
troponin C, cardiac/slow skeletal
Tnni1
troponin I, skeletal, slow 1
Tnnt3
troponin T3, skeletal, fast
Zic1
zinc finger protein of the cerebellum 1
absence of brown fat causes????
Thus, brown adipose tissue is essential for:
- classical nonshivering thermogenesis
- cold-acclimation recruited
adaptive adrenergic thermogenesis
-diet-adaptation recruited
adaptive adrenergic thermogenesis
(and without it, you get fat)
- all adaptive adrenergic thermogenesis?
- at least in mice!
-and likely in humans….
absence of brown fat causes????
mesenchymal
stem cell
white preadipocyte and
brite adipocyte
common precursor
Myf5
Hoxc9
Hoxc9
Tcf21
white adipocyte
Hoxc9
UCP1
”brite” adipocyte
PRDM16
miR-206
UCP1
adipomyocyte
and myocyte
common precursor
miR-206
myocyte
”adipomyocyte”
classical brown adipocyte
Evidence from man
UCP1 mRNA
levels
5
The -3826
polymorphism
determines
UCP1 expression
4
3
2
1
0
.
A/A
human intraperitoneal
adipose tisue
.
A/G
.
G/G
.
Based on data from
Esterbauer et al. 1998
Evidence from man
Correlation of UCP1 genotype with obesity
30
25
%
20
AG
GG
15
10
AA
5
0
1
2
3
4
Quartiles of BMI
Sramkova et al. 2007
Brown-fat cells:
20
Glucose uptake
15
10
5
0
NE + M
NE
0
.
.
ins + M
ins
0
.
Data from
Marette & Bukowiecki 1991
Would the presence/absence
of brown fat affect
”nonshivering thermogenesis”
Can adult humans demonstrate nonshivering thermogenesis?
A unique but clear result:
Cold-induced
nonshivering thermogenesis
in adult curarized man
***
0,2
nmol O
2
}
Nonshivering
thermogenesis
per kg
per min
0,1
Ar tificially ventilated,
electromygraphic monitored:
no shivering.
Incr ease in plasma noradrenaline
and plasma fatty acids
Adult man has NST
0
Control
Cold:
1 hour
- 14 °C
Jessen, Rabøl, W inkler 1980
160
160
140
140
· mg
-1
120
nmol O 2· min
100
80
60
Oleate
60 µM
40
120
100
Mit
80
60
20
0
Oleate
60 µM
GDP
40
Mit
0
2
3
Time, min
4
0
1
2
3
Time, min
4
Pyruvate
Palmitoyl-L-carnitine
GDP
160
Oleate, µM
140
160
140
-1
40
100
30
80
20
60
40
GDP
-1
· mg
50
120
Oleate, µM
70
60
-1
· mg
1
-1
0
nmol O 2· min
20
nmol O 2· min
· mg
-1
nmol O 2· min
When UCP1
is active
both fatty acids
and glucose
are oxidised
-1
GDP, Oleate
-1
Oleate
PLC
60
120
80
50
40
100
80
30
60
40
70
20
Pyr
20
20
0
0
0
2
4
6
Time, min
8
0
2
4
6
Time, min
8
Mice in the cold
Body temperature
40
60
Food consumption
Metabolism
8
6
30
40
g/mouse
ml O2/(min•kg0.75)
T b, °C
35
20
RMR
25
0
60
120
Time at 4°, min
180
2
0
0
20
4
0
10
20
30
Temperature, °C
0
10
20
30
Temperature, °C
40
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glucose disposal
FDG uptake
Heart
Colon
Brain
BAT
0
1
2
3
Relative uptake per tissue wet w eight
Rats, tissue extraction
Baba//Wahl 2007
4
FDG uptake
Cold-exposed
Heart
Colon
Brain
BAT
0
1
2
3
Relative uptake per tissue wet w eight
Rats, tissue extraction
Baba//Wahl 2007
4
FDG uptake
Cold-exposed
Heart
Colon
Brain
BAT
0
1
2
3
Relative uptake per tissue wet w eight
Rats, tissue extraction
Baba//Wahl 2007
4
metabolic syndrome
Retrospective study
Effect of temperature
7
%
brown-fat
positive
6
Same day temperature
5
4
3
2
1
0
-10
0
10
20
30
Outdoor temperature
Kim//Kim 2007
40
Dedicated study
Physiologically
regulated
active glucose uptake
brown adipose tissue
is found
in adult humans
warm-induced
obesity
The classical
philosophical question:
is the tree in the courtyard
still there
when we don’t see it?
And concerning differences in frequencies:
Do
women versus men
lean versus obese
more easily feel cold?
Are examination room temperatures
influenced by outside temperature?
(a few degrees make a difference)
Mice in the cold
Body temperature
40
60
Food consumption
Metabolism
8
6
30
40
g/mouse
ml O2/(min•kg0.75)
T b, °C
35
20
RMR
25
0
60
120
Time at 4°, min
180
2
0
0
20
4
0
10
20
30
Temperature, °C
0
10
20
30
Temperature, °C
40
Brown
fat
in
mice
and
men:
Active brown fat is found in (some) adult humans (but only visible when active)
Adrenergic thermogenesis in brown-fat cells is fully UCP1-dependent
Angiogenesis is stimulated by sympathetic nervous activity and is VEGFmediated. It is not hypoxia-dependent
Adaptive adrenergic thermogenesis (”diet-induced thermogenesis”)
is fully UCP1-dependent
No evidence (so far) for any thermogenic effects
of UCP2 or UCP3
Brown-fat and muscle cells derive (as energy-dissipating organs)
from a common progenitor cell - as do ”brite” and white-fat cells
Lack of UCP1 is sufficient to make (some) mice obese - at thermoneutrality
Lack of/low amount of UCP1 may be involved in (some) human obesities
Before 2007:
” in man, brown adipose tissue (UCP1)
is only found in newborns”