LR11 is a negative regulator of thermogenesis that antagonizes BMP signaling. LR11 knockout mice are resistant to diet-induced obesity and have increased energy expenditure and "browning" of white fat. LR11 regulates brown adipocyte oxygen consumption in vitro and its absence leaves the thermogenic program unchecked, increasing recruitment of brown adipocytes. Qualitative lipid alterations are important for adipose tissue function, and the adipose tissue expandability hypothesis links obesity to lipotoxicity from lipid spill over into other tissues when adipose capacity is exceeded.

1. University of Cambridge
Adipose tissue
expandability,
Lipotoxicity
and the
Metabolic
Syndrome
Toni Vidal-Puig
Institute of Metabolic Science
ajv22@cam.ac.uk
International Symposium
Latest in Obesity
Fundacion Ramon Areces

2. Mechanical Problems
Lipotoxicity
Metabolic Syndrome
Adipocentric view of the Metabolic Syndrome
• Fatty liver
• Diabetes
• Heart Failure
• Hypertension
• Dyslipidaemia
• Brain
• Macrophages
Aesthetic and Psychological
problems.
Metabolic problems
mismatch between energy
availability and
storage capacity
Obesity

3. Our research programme is focused on
understanding the link between obesity, insulin
resistance and cardiometabolic complications.
Our hypothesis is that failure in adipose tissue
expandability and functionality results in lipotoxicity.
We define lipotoxicity as ectopic accumulation of
lipids in organs other than adipose tissue and
believe that this ectopic lipid deposition is a major
mechanism linking obesity and metabolic
complications

4. Framework for research:
Theme 1. Improving Adipose tissue
expandability and function will
prevent/reverse ectopic lipotoxicity.
Theme 2 Promoting Energy dissipation.
Oxidising excess nutrients through different
strategies will prevent the accumulation of
toxic lipids.
Theme 3 Qualitative aspects of
lipotoxicity matters. Converting lipid
species to less toxic forms may prevent
metabolic complications in the context of
obesity.

5. Adipose tissue expandability hypothesis
(Virtue and Vidal-Puig. PLOS Bio(2008) BBA (Lipotoxicity issue)
a) The capacity to expand fat
mass to store lipid is a more
important determinant of
obesity associated metabolic
problems than
the absolute amount of
adipose tissue an individual
possesses.
b) Point of maximal
expansion determines fat
leakage and ectopic
storageleading to metabolic
complications.
LIPOTOXICITY
Adipose tissue expansion in not infinite
Lipid ‘spillover’
Adipokines
Caloric
excess

6. What factors may define adipose tissue expandability?
•Genetically determined number of preexisting preadipocytes
•Genetic programs of preadipocyte recruitment and adipogenesis
•Genetic programme of vasculogenesis/angiogenesis
•Dysfunction of other cellular components within the adipose tissue.
•Connective tissue/Extra cellular matrix
Connective tissue
Expansion
Vasculogenesis
Programme of
adipogenesis
Number of
pre-adipocytes
Caloric
excess and
associated
molecular
signals
Adipokine secretory
profile
Dynamics of fat cell turnover in humans. Spalding and Arner P Nature 2008.

7. Sethi & Vidal-Puig Chapter 3 In Metabolic Basis of Obesity 53-68 2011
Molecular players implicated in the transcriptional
regulation of adipogenesis
ADIPOGENESIS PROGRAMME

8. Local
autocrine and
paracrine
signals
regulate
progenitor
proliferation
and titrate
adipogenesis
Sethi JK, Diabetes 2010;59:2354-2357©2010 by American Diabetes Association
Christodoulides et al. J Cell Sci 2006
Lagathu et al. Diabetes, 2009
Lagathu et al. Int J Obes 2010
Xu et al. JBC, 1999
Sethi et al. FEBS Let. 2000
Christodoulides et al. Diabetologia
2006
Cawthorn et al. Cell Death Differ
2007
TRITATION ADIPOGENESS

9. Figure 23-4
Adhesion
Transmigration
Capture
Rolling
Integrins
Selectin
Ligands
(PSGL-1)
Monocytes
Selectins
(P/E-selectin)
V-CAM
I-CAM
Neutrophils
Lymphocytes
M1-like
macrophages
Activation
Chemokine
gradient
Chemokines
CCL(2,5)
CXCL(1,2,8)
Cytokines
IL-6
TNF-α
IL-1β
Activation
Adipocytes
Necrotic
adipocyte
PECAM-1
Differentiation
M2-like
macrophages
“M2”markers
CD206
Arg1
“M1”markers
CD11c
NOS2
Chemokine
Receptor
CCR(1-2-5)
CXCR(1-2)
Mast cells
ECs
Lymphocytes
Adipose tissue
inflammation

10. Obese ATMs are filled with
triglycerides & cholesterol
BODIPY F4/80 DAPI
16w ob/ob
Prieur X et al., Diabetes,
2011

11. Macrophage
Lipotoxicity
Prieur X, et al. Diabetes
60:797-809 (2011)
Fat spillover
Adiponectin
ARG1
EMC component
Vascularisation
Tissue remodeling
M
M2
M2
M2 M2
M1M2
Fat spillover
M1
M2
TNFa
M1
M2 TNFa
M2
M2 M2
Macrophage
Lipid droplets
M
M

12. The concept of metabolic set point
Epidemiologically the risk of diabetes increases linearly with increased
body weight.
Once an individual
reaches their maximal
adipose tissue mass, then
metabolic complications
ensue, suggesting that
individuals would go from
metabolically normal to
metabolically
compromised in a
relatively small weight
window.
Adipose tissue expandability hypothesis suggests this may not be the
case for an individual.

13. How the adipose tissue copes with expansion?
Allostasis applied to the mechanisms controlling membrane
lipid composition
Lipid composition of membranes – importance of phospholipids
Dynamic structures (growth, turnover, renewal)
Heterogeneous structures ( lipid rafts)
Communication Hubs for transduction pathways
•Compartmentalisation
•Signal transduction
•Cell adhesion
•Lipid traffic
•Ion channel function
•Receptor mobility
Need for mechanisms of quality
control

14. Lipidomics as a hypothesis generator tool
From Pietilainen PLOS Biology 2011

15. Research Article
Association of Lipidome Remodeling in the
Adipocyte Membrane with Acquired Obesity in
Humans
Pietiläinen KH, Róg T, Seppänen-Laakso T, Virtue S, Gopalacharyulu P, et
al. 2011 PLoS Biol 9(6): e1000623. doi:10.1371/journal.pbio.1000623

16. 22:4n-6
22:5n-6
18:4n-3
20:4n-3
14:0
16:0
18:0
20:0
16:1n-7
18:1
20:1
18:2n-6
18:3n-6
20:3n-6
20:4n-6
18:3n-3
20:5n-3
22:5n-3
22:6n-3
E
E
E
7
9 E
E
6
E
5
6
E
5
E
4
12:0
E
*
**
*
**
*
*
*
**
**
Non-essential fatty acids Essential fatty acids
**
Fatty acids Markers of
enzyme activities
*
*
A
B
Decreased
in obese co-twins
Increased
in non-obese co-twins
Changes in desaturation and elongation in adipose tissue of obese and
lean discordant twins
Unsaturation and elongation of fatty acids

17. • The fatty acid elongase Elovl6 is highly expressed in brown
adipose tissue (Moon et al., 2001).
• Elovl6 acts to convert C16 saturated and monounsaturated fatty
acids to C18 fatty acids and can potentially affect over 50% of the
cellular lipidome.
• Elovl6 product stearate has been implicated in the regulation of
mitochondrial function (Senyilmaz et al., 2015).
Elovl6 mediated fatty acid elongation;
• Hypothesis: Elovl6 may act to regulate
mitochondrial function and therefore thermogenesis
in BAT.

18. Decreased Maximal thermogenic
Capacity in ELovl6 KO at Low T
Decreased Energy Expenditure
in ELovl6 KO at Low T
Decreased NE
stimulated
glucose uptake
Decreased Gene expression analysis of electron transport chain complexes
Complex 1 Complex 2 Complex 3
Time
Virtue S, Cell Reports (in press) Tan et al Cell Reports (2015)

19. • Role for the elongation of non-essential 16-carbon fatty acids to 18
carbon fatty acids in the adaption of brown adipose tissue to cold.
• In physiological states (Ageing/TNHFD) where beiging of white adipose
tissue is prevented Elovl6 KO mice exhibit an impaired metabolic profile
• Ablation Elovl6, reduced overall maximal thermogenic capacity and led
to compensatory beiging of white adipose tissue depots.
• Mice lacking Elovl6 had lower brown adipose tissue thermogenic
capacity, which was associated with a reduction in expression on both an
mRNA and a protein level of components of the mitochondrial electron
transport chain.
Altogether these Data show
Tan et al Cell Reports (2015)

20. D Senyilmaz et al. Nature 000, 1-5 (2015) doi:10.1038/nature14601
Drosophila lacking Elovl6 and C18:0 have impaired
mitochondrial function.
Oxygen consumption
Survival

21. D Senyilmaz et al. Nature 000, 1-5 (2015) doi:10.1038/nature14601
C18:0 is required for mitochondrial fusion.
Fragmentation index

22. D Senyilmaz et al. Nature 000, 1-5 (2015) doi:10.1038/nature14601
C18:0 acts via TFR1, JNK and HUWE1 to regulate
mitofusin.
Lack of ELovl6
C18:0
Activation of TFR1
Activation of JNK
Phosphorylation of HUWE1
Ubiquitination MFN2
Mitochondria fragmentation
ELovl6
C18:0
Inactivation of TFR1
Inactivation of JNK
No Phosphorylation of HUWE1
No Ubiquitination MFN2
No Mitochondria fragmentation
Increased Fusion
GAMBOGIC Acid

23. Activating TFR1 with Gambogic in vivo directly
impairs thermogenic capacity
Control
Gambogic

24. Gambogic treatment reduces markers of thermogenesis and the
mitochondrial ETC in BAT and scWAT.
BAT

25. Brown fat activity modulators
Sanger Institute
TVPLab
LR11
BMP8b

26. Browning thermogenic activation
Adrenergic
receptors
NRP
receptor
FGF/KLB
receptors
Metabolic fuel
(lipid, glucose)
uptake/oxidation
Heat
Muscle Liver Heart Brain
FGF21
Natriuretic
peptides
Noradrenaline
SNS
Irisin
BMP8b
Preadipocytes BROWN/BEIGE ADIPOCYTE
Recent years there has been a burst in the identification of novel
molecules and pathways controlling BAT activity

27. Bone Morphogenetic Proteins (BMPs)
• Members of the TGF-
super family
• Secreted proteins
which bind type I and
type II membrane
receptors to regulate
transcription via
specific ‘Smad’
proteins.
LR11

28. BMP8b is enriched in mature brown adipocytes and
regulated by thermogenic stimuli
Tissue
distribution
BAT fractionation
BAT

29. BMP8b-/- mice have a reduced thermogenic
response to HFD-feeding
Whittle et al,
Cell, 2012
Body weights Energy
expenditure

30. NE-induced oxygen consumption in BMP8b-/-
acclimated to 4oC is reduced

31. “LR11 as a negative regulator of
thermogenesis”
Collaboration with Bujo lab at TOHO university

32. Background: LR11, an LDL receptor
• LDL receptor family
• Multifunctional receptors
• Cellular uptake of plasma lipoproteins – LDLR binds ApoB
and ApoE
• All members have 1-4 clusters of ligand binding repeats
Yamakazi et al, 1996
• Brain
• liver
• kidney
• WAT/BAT
• smooth muscle
cells
• Expression in brain
positively regulated
by docosahexenoic
acid (DHA) (Ma et al,
2007)
Tissue
distribution

33. Model for regulated intramembrane proteolysis of LR11 or SorLA.
Christopher Böhm et al. J. Biol. Chem. 2006;281:14547-
14553
©2006 by American Society for Biochemistry and Molecular Biology
Cell surface receptor
Metalloprotease
TACE/ADAM17
Soluble
form

34. Fig.3
Subcutaneous fat
Liver
Epididymal fat
Mesenteric fat Brown fat
Retroperitoneal fat
LR11+/+
LR11-/- LR11+/+ LR11-/- LR11+/+ LR11-/- LR11+/+ LR11-/-
LR11+/+ LR11-/-
LR11+/+ LR11-/-
LR11+/+ LR11-/-
LR11+/+ LR11-/-
D PHENOTYPING OF THE LR11 KO MOUSE
Whitle et al Nat Communication 2015

35. LR11-/- mice show resistance to diet-induced
obesity and have increased energy expenditure
Dr. Andrew Whittle and Dr. Meizi Jiang
Body weights Energy expenditure
Whitle et al Nat Communication 2015

36. “Browning” in LR11-/- subcutaneous white adipose tissue
H&E Whitle et al Nat Communication 2015

37. NE-induced oxygen consumption is increased
in HFD-fed LR11-/- mice raised at
thermoneutrality
Whitle et al Nat Communication 2015

38. LR11 regulates brown adipocyte oxygen
consumption in vitro
Dr. Andrew Whittle and Dr. Meizi Jiang
Primary brown adipocytes differentiated from the stromal vascular fraction of wild-type
or LR11-/- BAT, and from wild-type BAT treated with or without soluble LR11 (10
ng/mL) throughout differentiation, stimulated with NE (100 nM).
WT v. LR11-/-
brown adipocytes
Control v. sLR11 treated
wild-type brown adipocytes
functional consequences

39. LR11 antagonizes BMP signalling in White
adipocytes
LR11-/- white adipocytes have a greater increase in thermogenic gene expression
in response to BMP7 treatment. This response is blocked in both LR11-/- and wild-type
white adipocytes with sLR11 treatment.

40. Conclusions: LR11, a *negative* regulator of
thermogenesis.
• LR11 is a bona fide negative regulator of thermogenesis
• Its expression in adipose tissues with thermogenic potential
suggests it plays an important role in negatively regulating
thermogenesis, which if left unchecked could result in
hyperthermia or severe energy depletion.
• In the absence of LR11, as in LR11-/-, the thermogenic
programme is left unchecked, leading to increased
recruitment of brown adipocytes.

41. Conclusions
• Proper Adipose tissue function requires coordinated
interaction between adipocytes, precursors, immune cells,
blood vessels, nerves and ECM
• Obesity alters the ultrastructure and cellular composition of
WAT affecting its function and impairing the capacity to
buffer excess of nutrients.
• BAT could eliminate the excess of nutrients improving WAT
function
• Qualitative alterations of Lipids are important

42. Ana Pirraco Agnes Lukasik Martin Dale Stefania Carobbio Sergio Rodriguez-Cuenca
Katie Ketteridge-Lowe Crystal Mok Sam Virtue Toni Vidal-Puig Vivian Peirce Keli Phillips
Chong Yew Tan Mark Campbell Guillaume Bidault Maarten Soeters Vanessa Pellegrinelli Camilla Ingvorsen
Conall
Dennedy
TVPLab (@TVPLab) | Twitter
https://twitter.com/tvpla

43. Leo Krall
Jeff Flier
David Moller
Brad Lowell
Thilo Hagen
Chen Yu Zhang
Manuel Munoz
Serrano Rios
Patxi Sanchez Franco
Alberto Leiva
Jose Antonio Vazquez
Rafael Carmena
Gema Medina
Miguel Lopez
Nuria Barbarroja
Antonio Camargo
Joana Relat-Pardo
Hideaki Bujo
Rudy Zechner
Aurelio Teleman
Francesc Villarroya
Antonio Zorzano
Fernandez Real
Matej Oresic
Joaquin Dopazo
Juan Antonio Paniagua
Antonio Moschetta
Ulf Smith
Martin Brand
Lluis Fajas
Gemma Frubeck
Alessadro Bartolomucci
Diego Haro
Pedro Marrero
Barbara Cannon
Kamal Rahmuni
Carlos Dieguez
Manuel Ros
Mercedes Ricote
Thorkild Sorensen
Jackie Stephens
Andrew Goldberg
S O’Rahilly
Jasswinder Sethi
Jules Griffin
Barry Rosen
Andrew Whitle
Viv Pierce
Chris Lelliot
Alberto Camacho
Ishikawa K
Meirhaeghe A
Rachel Hagen
Romina Boiani
Costas christodulides
Nadeene Parker
Adrienne Kis
Claire Lagathu
Mark Slawick
Sarah Gray
Kiara Curtis
Ciaran Sewter
Edouardo de la Nora
Crystal Mock
Matthias Laudes
Sarawutt Jitrapakdee

44. IMS,
University of Cambridge
• Andrew Whittle
• Viv Pierce
• Stefania Carobbio
• Vanessa Pellegrinelli
• Sam Virtue
Dep of Biochemistry,
University of Cambridge
• Houjiang Zhou
• Kathryn Lilley
University of Cardiff
• Matthew White
• Alun Davies
University of Barcelona
• Joana Relat
WGI
• Barbara Cannon
Toho University
• Hideaki Bujo
Chiba University
• Meizi Jiang
• Wolfgang Schneider
Acknowledgements
Sanger Institute
• Chris Lelliott
• Camilla IvrognsenUIOWA
Kamal Rahmouni
Donald Morgan
USC
Miguel Lopez
Luis Martins
Rosalia Gallego
Medical Research Council
British Heart Foundation
Wellcome
ERC
EU-FP7 Etherpath
2020 BetaBat
2020 EpooS

45. Cherry Hinton Lions TVPLab