Mechanisms driving adipose tissue mass growth

-the role of fat cell size and local inflammation

Mikael Rydén M.D, Ph.D

Karolinska Institutet Dep of Medicine

Karolinska University Hospital, Huddinge Stockholm Sweden

Obesity development-the old view Once generated in childhood, fat cells are retained and alter in size according to weight changes Based on studies of fat cell size and number on prisoners before and after dietary induced weight change

Obesity development-the new view The growth of fat tissue is dependent on a constant renewal (adipogenesis) involving both cell growth (hypertrophy) and proliferation (hyperplasia)

Why?.....

14 C reading Cellular

age

Strategy to establish cellular age by measuring the nuclear bomb test derived 14C in DNA

Lipid filling

Cell death

Adipocyte turnover in adult humans •10% of the adipocyte pool turned-over each year •Rate of adipogenesis and cell death 2-fold increased in obesity

Lipid filling

Cell death

obese nonobese Spalding et al Nature 2008;453:783-7

0

1

2

3

4

5

6

7

8

9

0.5 3 9 15 35 55 60+

A g e

Fat c

ell n

umbe

r x

10

10

Constant fat cell number during ageing among adults

obese

lean Children data from Knittle et al JCI 63:239,1979

Spalding et al Nature 453:783,2008

Adipocyte turnover in adult humans Fat cell number is constant!

•Constant fat cell number throughout adult life Spalding et al Nature 2008;453:783-7 •What about weight loss?

Bariatric surgery Spalding et al Nature 2008;453:783-7

Cancer cachexia Rydén et al Cancer 2008 113:1695-704

Relationship between fat cell size and body fat

0

300

600

900

1200

1500

Fat

cel

l vou

me,

pl

0 20 40 60 80 100 120 140 Body fat, kg

Large fat cells associate with •Insulin resistance •Increased risk of developing type 2 diabetes

HOW CAN WE SEPARATE THE

EFFECT OF CELL SIZE

FROM THE EFFECT OF BODY FAT?

How can we estimate the impact of fat cell size? Algorithm to obtain body fat independent classification

0

300

600

900

1200

1500

Fat

cel

lvo

ume,

pl

0 20 40 60 80 100 120 140 Body fat, kg

Hypertrophy

Found-expected volume (+value) = degree of hypertrophy

Hyperplasia

Found-expected volume (-value) = degree of hyperplasia

E Arner et al Diabetes, 59: 17-25, 2010

”Morphology value”

Hypertrophy

Hyperplasia

0 5

10 15 20 25 30 35 40 45

BM

I

Obese 0

1

2

3

4

5

6

HO

MA

p<0.001

p<0.001

Lean

Insulin resistance Body mass index

Relationship between the morphology value and insulin sensitivity in 230 lean and 400 obese

Lean Obese

Obese

p<0.01

p=0.01

Lean

Insulin stimulated Basal (spontaneous)

Adipose cellularity in 90 lean and 310 obese Adipocyte lipogenesis

Lean Obese 0

1

2

3

4

Lip

ogen

esis

per

10

mill

ion

cells

0

2

4

6

8

10

12

Lip

ogen

esis

per

10

mill

ion

cells

NS NS

Hypertrophy

Hyperplasia

Noradrenaline-stimulated Basal (spontaneous)

Adipose cellularity in 220 lean and 300 obese women Adipocyte lipolysis

Lip

olys

is p

er 1

0 m

illio

n ce

lls

0

3

6

9

p<0.0001

p<0.0001

Lip

olys

is p

er 1

0 m

illio

n ce

lls

Lean Obese 0

3

6

9

12

Lean Obese

NS NS

Hypertrophy

Hyperplasia

HYPERPLASIA HYPERTROPHY

Fat cell size and number matters

•Risk to develop type 2 diabetes C Weyer et al Diabetologia 43: 1498-1506, 2000 M Lönn et al FASEB J. 24:326-31. 2010 •Strong heredity for type 2 diabetes P Arner et al PLoS One 6:e18284, 2011

Protective

Irrespective of whether you are lean or obese, your adipose tissue can be in two different ways

Much lipid filling

Cell death

Mechanisms causing different forms of morphology

Less lipid

filling

Cell death

SLOW TURNOVER

⇓ hypertrophy

RAPID TURNOVER ⇓

hyperplasia E Arner et al Diabetes, 59:105-9, 2010

WHAT KEEPS HUMAN ADIPOCYTE TURNOVER GOING?

The old view

0

200

400

600

800

Sec

retio

n pe

r tis

sue

w

ei g

ht

*

TNF-alpha IL-6 MCP-1

The inflamed adipose tissue among obese

Secretion of inflammatory proteins from human sc

adipose tissue

NONOBESE OBESE

Leucocyte infiltration in obese adipose tissue

GREEN = the leukocyte antigen CD45 BLUE = nuclear staining with DAPI

*

* * *

*

*

*

0

2 4

6 8

10 12

14

Lean Subc obesity

Visceral obesity

Subc Omental

Macrophages in human adipose tissue.

Percentage of all tissue cells

Adopted from Harman-Boehm et al, JCEM, 92:2240-47, 2006

Macrophage infiltration and TNFα secretion in adipose tissue obese and nonobese subjects

0

1

2

3

4

5

6

Lean n=25

Obese n=100

50%

TNFα secretion from sc adipose tissue

Rydén M et al, unpublished

Effects of TNFα on adipocytes

• Increases lipolysis • Decreases lipogenesis • Attenuates preadipocyte proliferation and

adipocyte differentiation • Increases adipocyte cell death

Reviewed in Rydén M and Arner P. J Intern Med. 200 Oct;262(4):431-8.

Are adipose inflammatory factors

just bad or have we

misunderstood them?

Adipokine secretion in relation to adipose cellularity in lean healthy women (BMI<25 kg/m2)

E Arner et al N Engl J Med, 362:1151-3, 2010 T

NF-

alph

a se

cret

ion

0

200

400

600

800

1000

1200

-400 -300 -200 -100 0 100 200

Adipose morphology, picolitres

r2 = 0.32 p = 0.005

0

100

200

300

400

500

600

-400 -300 -200 -100 0 100 200 Adipose morphology, picolitres

Il-6

sec

retio

n A

dipo

nect

in s

ecre

tion

0

100

200

300

400

-400 -300 -200 -100 0 100 200

Adipose morphology, picolitres

0

2

4

6

8

10

12

-400 -300 -200 -100 0 100 200

Adipose morphology, picolitres

Lep

tin s

ecre

tion

0

200

400

600

800

1000

1200

-0.1 0.2 0.5 0.8 1.1 1.4 1.7 2.0

TN

F-al

pha

secr

etio

n r2 = 0.43 p = 0.0018

0

200

400

600

800

1000

1200

0 0.5 1.0 1.5 2.0 2.5

TNF-alpha mRNA

r2 = 0.38 p = 0.005

TNF-alpha mRNA

TN

F-al

pha

secr

etio

n

Tissue Adipocytes

TNF-alpha secretion and gene expression in adipose tissue of

lean healthy women (BMI<25 kg/m2) E Arner et al N Engl J Med, 362:1151-3, 2010

Could TNFα play a physiological role?

• Inhibit adipocyte growth? • Limit fat cell proliferation and increase fat

cell death? • A response which becomes maladaptive in

an environment with constant caloric over-supply?

Upstream regulator(s) of human adipose

inflammation?

TF

TF TF

TF TF

TF

TF

TF

TF TF

MCP-1 TNF-α IL-1

A network of transcription factors is linked to inflammatory proteins in human fat cells

Twist-1 • In a gene expression profile on sc adipose tissue from lean and

obese twist1 expression was significantly lower in obese subjects (Dahlman I unpublished data)

• Basic helix-loop-helix transcription factor • Identified in Drosophila melanogaster • Binds to E-boxes with the CANNTG motif • Implicated in bone formation and tumor metastasis • Mutations associated with Saethre-Chotzen syndrome • Twist-1 and -2, 66% identity at aa-level • Twist2-/- and twist1+/- twist2+/- mice have increased levels of

TNFα,IL-6 and IL-1β (Šošić D et al. Cell 2003)

• Twist1 negatively regulates transcriptional activity of PGC-1α in murine BAT (Pan D et al. Cell 2009)

Relationship between Twist-1 gene expression and secretion of inflammatory proteins in human subcutaneous adipose tissue

A. B. IL

-6 s

ecre

tion/

cel

l

low twist-1

high twist-1

*

0 2 4 6 8

10 12 14

TNFα

sec

retio

n / c

ell

0 0.5

1 1.5

2 2.5

3 3.5

4

*

low twist-1

high twist-1

Petterson A et al, J Clin Endocrinol Metab, 96:133-41, 2010

0

100

200

300

400

Rel

ativ

e lu

cife

rase

act

ivity

(f

old

indu

ctiu

on)

pIL6 +pcDNA3.1

pIL6 +Twist1

*

Luciferase

-541 +68 pIL6

Twist1 regulates cytokine expression 3T3-L1 cells were transfected with Twist1 cDNA and a gene reporter

construct for IL-6 (pIL) or empty pcDNA3.1

Petterson A et al, Diabetes, 59:564-71, 2010

exon exon exon promotor

mRNA ⇓

Production of TNF-α, Il-6 and MCP-1

Interaction between Twist-1 and inflammation in human adipose tissue

Twist-1

Petterson A et al, Diabetes, 59:564-71, 2010

Reduced Twist1 potentiates the pro-inflammatory effect of TNFα

Petterson A et al, J Clin Endocrinol Metab, 96:133-41, 2010

Relationship between Twist-1 gene expression and the morphology of

human subcutaneous adipose tissue

Petterson A et al, J Clin Endocrinol Metab, 96:133-41, 2010

Hyperpl.

Hypertr.

Hyperpl.

Hypertr.

0

20

40

60

80

Twis

t1 m

RN

A (A

.U.)

Non-obese Obese

F= 21.3 p < 0.001

Glucose

Role of adipose inflammation in man

FFA

Mesenchymal stem cells

TNFα

Macrophages

MCP-1 TNFα

DIFFERENTIATION AND LIPID FILLING OF FAT CELLS

Lipids *

TNFα

Pre-adipocytes

TNFα

CELL DEATH

TNFα

hyperplasia hypertrophy

Protected

Adipose cellularity is clinically important for nonobese

Decreased insulin sensitivity

Type 2 diabetes risk

Alterations in transcription

factor regulation

SUMMARY The link between inflammation and adipogenesis

•There is a high turnover of fat cells in adult life although the total number of fat cells is constant

•Hypertrophic fat (few but large cells) is linked to development of insulin resistance irrespective of BMI

•Lean apparently healthy subjects with adipose hypertrophy have decreased insulin sensitivity

•Adipocyte turnover is an important determinant of adipose hypertrophy/hyperplasia, turnover (and thereby adipogenesis) and is high in hyperplasia

•Local inflammation may impact on morphology (hypertrophy/hyperplasia) of adipose tissue, hypertrophy is associated with inflammation

•Adipose inflammation is governed by a network of transcription factors

•Characterization of factors regulating turnover/morphology currently under way

LIPID LABORATORY •Peter Arner •Gaby Åström •Eva Sjölin •Kerstin Wåhlen •Clara Bambace •Amanda Petterson •Ingrid Dahlman •Britt-Marie Leijonhufvud •Katarina Hertel •Hans Wahrenberg •Elisabeth Dungner •Jurga Laurencikiene •Johan Hoffstedt •Patrik Löfgren •Daniel Andersson •Agne Kulyte •Silvia Lorente •Niklas Mejhert

•Erik Arner •Lennart Blomqvist •Kirsty Spalding •Pål Westermark •Samuel Bernard •Bruce Bucholz •Olaf Bergman •Tom Britton •Anneli Attersand •Pauline Decaunes •Anne Bouloumie •Carsten Daub •Karine Clement •Nicolas Veneteclef •Mats Eriksson

ADIPOCYTE TURNOVER WORKING FORCE EXTERNAL COLLABORATION

•Hernan Concha •Moustapha Hassan •Jonas Frisén •Erik Näslund •Per Heden •Claes Carnheim •Ian Cassady •David Hume •Shalender Bhasin •Karin Dahlman-Wright •Mireille Cormont •Jean-Francois Tanti •Erik van Nimwegen •Keith Frayn

Funded by 6:th and 7:th EU framework NUGENOB, Cost Action BM0602, ADAPT

Thank you for your attention!