University of Cambridge

Adipose tissue expandability,Lipotoxicity

and the Metabolic Syndrome

Toni Vidal-PuigInstitute of Metabolic Scienceajv22@cam.ac.uk

International Symposium Latest in ObesityFundacion Ramon Areces

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 problemsmismatch between energy availability and storage capacity

Obesity

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

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.

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 thanthe 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

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 tissueExpansion

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.

Sethi & Vidal-Puig Chapter 3 In Metabolic Basis of Obesity 53-68 2011

Molecular players implicated in the transcriptional regulation of adipogenesis

ADIPOGENESIS PROGRAMME

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 2006Lagathu et al. Diabetes, 2009Lagathu et al. Int J Obes 2010

Xu et al. JBC, 1999Sethi et al. FEBS Let. 2000Christodoulides et al. Diabetologia 2006Cawthorn et al. Cell Death Differ 2007

TRITATION ADIPOGENESS

Figure 23-4

AdhesionTransmigration

CaptureRolling

IntegrinsSelectin Ligands(PSGL-1)

Monocytes

Selectins(P/E-selectin)

V-CAMI-CAM

NeutrophilsLymphocytes

M1-likemacrophages

Activation

Chemokinegradient

ChemokinesCCL(2,5)

CXCL(1,2,8)

CytokinesIL-6

TNF-αIL-1β

ActivationAdipocytes

Necrotic adipocyte

PECAM-1Differentiation

M2-likemacrophages

“M2”markersCD206Arg1

“M1”markersCD11cNOS2

ChemokineReceptor

CCR(1-2-5)CXCR(1-2)

Mast cells

ECs

Lymphocytes

Adipose tissue inflammation

Obese ATMs are filled with triglycerides & cholesterol

BODIPY F4/80 DAPI 16w ob/ob

Prieur X et al., Diabetes, 2011

Macrophage Lipotoxicity

Prieur X, et al. Diabetes 60:797-809 (2011)

Fat spillover

Adiponectin

ARG1

EMC componentVascularisationTissue remodeling

M

M2

M2

M2 M2

M1M2

Fat spilloverM1

M2TNFa

M1

M2 TNFaM2

M2 M2

MacrophageLipid droplets

MM

The concept of metabolic set pointEpidemiologically 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.

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

Lipidomics as a hypothesis generator tool

From Pietilainen PLOS Biology 2011

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

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

12:0E

*

**

*

**

*

*

*

**

**

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

• 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.

Decreased Maximal thermogenicCapacity 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)

• 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)

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

D Senyilmaz et al. Nature 000, 1-5 (2015) doi:10.1038/nature14601

C18:0 is required for mitochondrial fusion.

Fragmentation index

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 fragmentationIncreased Fusion

GAMBOGIC Acid

Activating TFR1 with Gambogic in vivo directly impairs thermogenic capacity

Control

Gambogic

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

BAT

Brown fat activity modulators

Sanger Institute

TVPLab

LR11

BMP8b

Browning thermogenic activation

Adrenergicreceptors

NRPreceptor

FGF/KLBreceptors

Metabolic fuel(lipid, glucose)

uptake/oxidation

Heat

Muscle Liver Heart Brain

FGF21Natriuretic 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

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

BMP8b is enriched in mature brown adipocytes and regulated by thermogenic stimuli

Tissue distribution

BAT fractionation

BAT

BMP8b-/- mice have a reduced thermogenic response to HFD-feeding

Whittle et al, Cell, 2012

Body weights Energy expenditure

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

“LR11 as a negative regulator of thermogenesis”

Collaboration with Bujo lab at TOHO university

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

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

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

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

“Browning” in LR11-/- subcutaneous white adipose tissue

H&E Whitle et al Nat Communication 2015

NE-induced oxygen consumption is increased in HFD-fed LR11-/- mice raised at thermoneutrality

Whitle et al Nat Communication 2015

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 adipocytesControl v. sLR11 treated

wild-type brown adipocytes

functional consequences

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.

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.

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

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) | Twitterhttps://twitter.com/tvpla

Leo KrallJeff FlierDavid MollerBrad LowellThilo HagenChen Yu Zhang

Manuel MunozSerrano RiosPatxi Sanchez FrancoAlberto LeivaJose Antonio VazquezRafael Carmena

Gema MedinaMiguel LopezNuria BarbarrojaAntonio CamargoJoana Relat-Pardo

Hideaki BujoRudy ZechnerAurelio TelemanFrancesc VillarroyaAntonio ZorzanoFernandez RealMatej OresicJoaquin DopazoJuan Antonio PaniaguaAntonio MoschettaUlf SmithMartin BrandLluis FajasGemma FrubeckAlessadro BartolomucciDiego HaroPedro MarreroBarbara CannonKamal RahmuniCarlos DieguezManuel RosMercedes RicoteThorkild SorensenJackie StephensAndrew Goldberg

S O’RahillyJasswinder SethiJules GriffinBarry Rosen

Andrew WhitleViv PierceChris LelliotAlberto CamachoIshikawa KMeirhaeghe ARachel HagenRomina BoianiCostas christodulidesNadeene ParkerAdrienne KisClaire LagathuMark SlawickSarah GrayKiara CurtisCiaran SewterEdouardo de la NoraCrystal MockMatthias LaudesSarawutt Jitrapakdee

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 RelatWGI• Barbara Cannon

Toho University• Hideaki Bujo

Chiba University• Meizi Jiang• Wolfgang Schneider

Acknowledgements

Sanger Institute

• Chris Lelliott• Camilla Ivrognsen UIOWA

Kamal RahmouniDonald Morgan

USCMiguel LopezLuis MartinsRosalia Gallego

Medical Research CouncilBritish Heart FoundationWellcomeERC EU-FP7 Etherpath2020 BetaBat2020 EpooS

Cherry Hinton Lions TVPLab