Dysregulation

of

the

expression

and

secretion

of

inflammation-related

adipokines

by

hypoxia

in

human

adipocytes

The

effect

of

hypoxia

induced

by

incubation

under

low

-LRB-

-RRB-

oxygen

tension

or

by

exposure

to

on

the

expression

and

secretion

of

inflammation-related

adipokines

was

examined

in

human

adipocytes

Hypoxia

led

to

a

rapid

and

substantial

increase

-LRB-

greater

than

sevenfold

by

h

of

exposure

to

-RRB-

in

the

hypoxia-sensitive

transcription

factor

in

human

adipocytes

This

was

accompanied

by

a

major

increase

-LRB-

up

to

-RRB-

in

transporter

mRNA

level

Hypoxia

-LRB-

or

-RRB-

led

to

a

reduction

-LRB-

up

to

threefold

over

h

-RRB-

in

adiponectin

and

haptoglobin

mRNA

levels

adiponectin

secretion

also

decreased

No

changes

were

observed

in

TNFα

expression

In

contrast

hypoxia

resulted

in

substantial

increases

in

FIAF/angiopoietin-like

protein

leptin

MIF

and

vascular

endothelial

growth

factor

-LRB-

VEGF

-RRB-

mRNA

levels

The

largest

increases

were

with

FIAF

-LRB-

maximum

-RRB-

leptin

-LRB-

maximum

-RRB-

and

VEGF

-LRB-

maximum

-RRB-

these

were

reversed

on

return

to

normoxia

The

secretion

of

leptin

MIF

and

VEGF

from

the

adipocytes

was

also

stimulated

by

exposure

to

These

results

demonstrate

that

hypoxia

induces

extensive

changes

in

human

adipocytes

in

the

expression

and

release

of

inflammation-related

adipokines

Hypoxia

may

underlie

the

development

of

the

inflammatory

response

in

adipocytes

leading

to

obesity-associated

diseases

Introduction

White

adipose

tissue

has

traditionally

been

considered

as

primarily

a

vehicle

for

the

storage

of

fuel

in

the

form

of

triacylglycerols

but

several

other

functions

are

now

recognised

for

the

tissue

In

particular

adipose

tissue

is

a

major

endocrine

organ

secreting

several

key

hormones

notably

leptin

and

adiponectin

-LSB-

-RSB-

These

hormones

are

part

of

the

large

group

of

protein

signals

and

factors

secreted

by

adipocytes

termed

adipokines

many

of

which

are

linked

to

immunity

and

the

inflammatory

response

-LSB-

-RSB-

These

encompass

major

cytokines

and

chemokines

including

TNFα

and

MIF

-LSB-

-RSB-

Obesity

which

is

characterised

by

a

major

expansion

in

adipose

tissue

mass

is

associated

with

a

state

of

chronic

mild

inflammation

there

being

increased

circulating

levels

of

inflammatory

markers

such

as

C-reactive

protein

haptoglobin

MIF

and

-LSB-

-RSB-

The

production

of

a

number

adipokines

increases

during

the

expansion

of

adipose

tissue

mass

in

the

obese

-LRB-

for

example

leptin

MIF

TNFα

and

-RRB-

with

the

result

that

there

is

an

inflammatory

state

within

the

tissue

-LSB-

-RSB-

An

exception

is

adiponectin

which

has

an

anti-inflammatory

action

-LSB-

-RSB-

Inflammation

in

WAT

is

considered

to

be

causal

in

the

development

of

type

diabetes

and

the

metabolic

syndrome

linked

to

obesity

-LSB-

-RSB-

There

has

however

been

little

focus

on

why

the

rise

in

adipose

tissue

mass

in

the

obese

should

lead

to

the

increased

production

of

inflammatory

adipokines

One

possibility

which

we

have

recently

suggested

is

that

it

is

a

response

to

relative

hypoxia

in

clusters

of

adipocytes

distant

from

the

vasculature

inflammation

serving

to

increase

blood

flow

and

stimulate

angiogenesis

-LSB-

-RSB-

Hypoxia

which

occurs

for

example

in

solid

tumours

and

during

wound

healing

induces

a

series

of

adaptive

responses

by

cells

and

a

key

role

in

the

transmission

of

the

hypoxic

response

is

played

by

the

hypoxia-inducible

transcription

factors

particularly

hypoxia-inducible

-LRB-

-RRB-

-LSB-

-RSB-

is

composed

of

two

subunits

which

is

constitutively

expressed

and

which

is

recruited

in

response

to

low

tension

to

yield

the

functional

transcription

factor

-LSB-

-RSB-

is

present

in

murine

clonal

adipocytes

and

hypoxia

has

been

shown

to

induce

an

increase

in

the

expression

of

leptin

and

vascular

endothelial

growth

factor

-LRB-

VEGF

-RRB-

in

these

cells

-LSB-

-RSB-

Recently

hypoxia

has

also

been

reported

to

induce

the

production

of

and

to

inhibit

the

synthesis

of

adiponectin

by

adipocytes

-LSB-

-RSB-

it

is

also

reported

to

induce

the

expression

of

visfatin

in

these

cells

-LSB-

-RSB-

has

been

identified

in

human

adipose

tissue

and

is

reported

to

be

increased

in

obesity

-LSB-

-RSB-

However

the

extent

to

which

human

adipocytes

respond

to

hypoxia

and

which

genes

are

hypoxia-sensitive

are

unknown

In

the

present

study

we

have

examined

the

effects

of

hypoxia

on

the

expression

and

secretion

of

key

adipokines

linked

to

inflammation

in

human

adipocytes

differentiated

from

fibroblastic

preadipocytes

in

culture

The

results

demonstrate

that

hypoxia

whether

through

low

tension

or

induced

chemically

by

leads

to

a

marked

recruitment

of

in

human

adipocytes

and

that

expression

of

the

facilitative

glucose

transporter

in

adipocytes

is

hypoxia-sensitive

Importantly

the

expression

and

secretion

of

several

adipokines

including

FIAF/angiopoietin-like

protein

leptin

MIF

and

VEGF

is

stimulated

by

hypoxia

in

human

adipocytes

while

adiponectin

and

haptoglobin

are

inhibited

Materials

and

methods

Human

adipocyte

culture

Human

subcutaneous

preadipocytes

derived

from

adipose

tissue

pooled

from

seven

female

subjects

were

obtained

-LRB-

together

with

culture

media

-RRB-

from

Zen-Bio

-LRB-

USA

-RRB-

The

patients

had

a

mean

body

mass

index

of

-LRB-

range

-RRB-

and

average

age

of

years

-LRB-

range

years

-RRB-

Cells

were

trypsinized

from

a

flask

and

plated

at

a

density

of

in

a

plate

and

maintained

in

preadipocyte

medium

containing

Dulbecco

's

modified

Eagle

's

medium

-LRB-

DMEM

-RRB-

Ham

's

-LRB-

v/v

-RRB-

fetal

calf

serum

-LRB-

FCS

-RRB-

mM

HEPES

U/ml

penicillin

μg/ml

streptomycin

and

μg/ml

amphotericin

B

at

°C

in

a

humidified

atmosphere

of

Cells

were

induced

at

confluence

by

incubation

in

differentiation

medium

composed

of

adipose

medium

-LRB-

AM

-RRB-

supplemented

with

mM

isobutyl

methylxanthine

and

μM

of

a

PPARγ

agonist

for

days

The

cells

were

then

cultured

with

AM

containing

DMEM/Ham

's

-LRB-

v/v

-RRB-

FCS

μM

dexamethasone

U/ml

penicillin

μg/ml

streptomycin

and

μg/ml

amphotericin

B

The

medium

was

changed

every

days

Fully

differentiated

cells

at

day

post-induction

were

treated

with

or

exposed

to

for

up

to

h.

For

treatment

wells

incubated

without

were

used

as

controls

μM

was

used

in

initial

experiments

and

μM

was

employed

in

a

time

course

study

For

exposure

to

hypoxia

the

cells

were

transferred

to

a

modular

incubator

chamber

-LRB-

Billups-Rosenberg

USA

-RRB-

which

was

flushed

with

and

and

sealed

and

placed

at

°C

for

up

to

h

as

indicated

The

control

cells

were

cultured

in

a

standard

incubator

-LRB-

and

-RRB-

The

cells

were

harvested

in

μl

of

Trizol

-LRB-

Invitrogen

UK

-RRB-

or

μl

of

lysis

buffer

per

well

at

the

stated

time

points

Media

were

collected

and

centrifuged

at

rpm

for

min

to

remove

cell

debris

and

the

supernatant

stored

at

°C

until

required

for

analysis

All

incubations

at

each

time

point

were

performed

in

replicates

of

up

to

six

wells

RNA

extraction

and

cDNA

synthesis

Total

RNA

was

isolated

from

cells

using

Trizol

and

μg

of

RNA

was

treated

with

DNase

I

-LRB-

Invitrogen

-RRB-

according

to

the

manufacturer

's

instructions

RNA

concentration

was

quantified

from

the

absorbance

at

nm

all

samples

had

a

nm

absorbance

ratio

of

One

microgram

of

DNase

I-treated

RNA

was

reverse-transcribed

using

a

Reverse-iT

strand

synthesis

kit

-LRB-

Abgene

UK

-RRB-

in

the

presence

of

anchored

oligo

dT

in

a

total

volume

of

μl

Real-time

PCR

Quantitative

real-time

polymerase

chain

reactions

-LRB-

PCRs

-RRB-

were

carried

out

in

a

final

volume

of

μl

consisting

of

ng

of

reverse-transcribed

cDNA

mixed

with

optimal

concentrations

of

primers

and

probe

and

qPCR

core

kit

-LRB-

Eurogentec

UK

-RRB-

in

plates

on

a

detector

-LRB-

Stratagene

USA

-RRB-

The

primer

and

probe

sets

were

designed

using

Primer

Express

software

-LRB-

Applied

Biosystems

-RRB-

and

synthesized

commercially

the

primers

and

TaqMan

probes

were

from

Eurogentec

TaqMan

probes

were

labelled

with

a

reporter

fluorescent

dye

-LRB-

FAM

-RRB-

at

the

end

and

a

fluorescent

dye

quencher

-LRB-

TAMRA

-RRB-

at

the

end

The

sequence

and

optimal

concentrations

of

primers

and

probes

together

with

the

size

of

products

are

as

detailed

previously

-LSB-

-RSB-

with

the

exception

of

FIAF

MIF

and

which

were

as

follows

-LRB-

bp

-RRB-

-LRB-

forward

-RRB-

-LRB-

reverse

-RRB-

and

FAM-TGGAGCAGCTACCCTGGATGTCCTATCTGA-TAMRA-3

-LRB-

probe

-RRB-

FIAF

-LRB-

bp

-RRB-

-LRB-

forward

-RRB-

-LRB-

reverse

-RRB-

and

-LRB-

probe

-RRB-

-LRB-

bp

-RRB-

-LRB-

forward

-RRB-

-LRB-

reverse

-RRB-

and

-LRB-

probe

-RRB-

MIF

-LRB-

bp

-RRB-

-LRB-

forward

-RRB-

-LRB-

reverse

-RRB-

and

-LRB-

probe

-RRB-

-LRB-

bp

-RRB-

-LRB-

forward

-RRB-

-LRB-

reverse

-RRB-

and

FAM-TGCTGGACCCACCGGCATGTTC

-LRB-

probe

-RRB-

Typically

the

amplification

started

with

min

at

°C

min

at

°C

and

then

cycles

of

the

following

s

at

°C

and

min

at

°C

Human

was

used

as

an

endogenous

reference

This

housekeeping

gene

was

selected

based

on

comparison

with

β-actin

where

demonstrated

no

significant

changes

in

expression

when

exposed

to

and

-LRB-

data

not

shown

-RRB-

Relative

quantitation

values

were

expressed

using

the

method

-LRB-

see

user

bulletin

no.

ABI

Prism

pp

Applied

Biosystems

-RRB-

as

fold

changes

in

the

target

gene

normalised

to

the

reference

gene

-LRB-

-RRB-

and

related

to

the

expression

of

the

untreated

controls

The

PCR

efficiency

in

all

runs

was

close

to

and

all

samples

were

analysed

in

at

least

duplicate

Measurement

of

and

adipokines

by

ELISA

Total

in

cell

lysates

was

measured

with

an

enzyme-linked

immunosorbent

assay

-LRB-

ELISA

-RRB-

kit

-LRB-

R&D

Systems

UK

-RRB-

according

to

the

manufacturer

's

protocol

Cells

were

solubilised

in

lysis

buffer

consisting

of

mM

Tris

-LRB-

pH

-RRB-

mM

NaCl

-LRB-

w/v

-RRB-

glycerol

mM

ethylenediaminetetraacetic

acid

mM

mM

β-glycerophosphosphate

mM

NaF

Triton

μg/ml

leupeptin

μg/ml

pepstatin

and

μg/ml

aprotinin

Before

measuring

by

ELISA

the

total

protein

content

of

the

lysates

was

determined

by

the

BCA

protein

assay

reagent

-LRB-

Sigma

UK

-RRB-

Adiponectin

leptin

MIF

and

VEGF

were

measured

in

cell

culture

media

using

commercial

ELISA

kits

-LRB-

R&D

Systems

-RRB-

The

assays

were

conducted

in

microplates

according

to

the

manufacturer

's

instructions

Measurement

of

by

Western

blotting

Samples

were

prepared

as

described

above

for

the

ELISA

assay

Fifteen

micrograms

of

protein

were

separated

by

sodium

dodecyl

sulfate

polyacrylamide

gel

electrophoresis

and

then

transferred

to

a

nitrocellulose

membrane

The

membranes

were

blocked

and

probed

with

polyclonal

goat

anti-human

-LRB-

R&D

Systems

-RRB-

or

mouse

monoclonal

anti-α-tubulin

-LRB-

Sigma

UK

-RRB-

as

the

primary

antibody

then

subjected

to

HRP-conjugated

anti-goat

IgG

-LRB-

R&D

systems

-RRB-

or

anti-mouse

IgG

-LRB-

Santa

Cruz

Biotechnology

-RRB-

as

the

secondary

antibody

Specific

proteins

were

visualised

with

the

enhanced

chemiluminescence

reagent

-LRB-

Amersham

UK

-RRB-

Statistical

analysis

The

results

are

expressed

as

mean

values

SE

Differences

between

groups

were

analysed

by

unpaired

two-tailed

Student

's

t

tests

Results

expression

during

differentiation

of

human

adipocytes

We

first

investigated

whether

human

white

adipocytes

express

the

gene

and

synthesize

the

encoded

protein

when

differentiated

in

culture

Quantitative

changes

in

gene

expression

after

the

induction

of

adipocyte

differentiation

were

analysed

by

real-time

PCR

Although

the

gene

was

expressed

both

before

and

after

differentiation

there

was

a

marked

decrease

in

mRNA

level

after

differentiation

was

induced

-LRB-

Fig.

-RRB-

By

day

the

mRNA

level

fell

to

of

that

seen

at

day

and

remained

low

The

expression

of

a

classical

adipokine

leptin

was

determined

as

a

reference

gene

As

expected

leptin

mRNA

was

differentiation-dependent

and

detectable

only

at

day

after

the

induction

of

differentiation

increasing

progressively

thereafter

-LRB-

Fig.

-RRB-

Fig.

course

of

quantitative

changes

in

mRNA

level

-LRB-

a

-RRB-

leptin

mRNA

level

-LRB-

b

-RRB-

and

protein

-LRB-

c

-RRB-

during

the

differentiation

and

development

of

human

adipocytes

Confluent

human

preadipocytes

-LRB-

day

-RRB-

were

differentiated

and

cultured

for

up

to

days

Relative

mRNA

levels

were

normalised

to

human

at

day

-LRB-

or

day

when

no

signal

was

evident

at

day

-RRB-

Total

protein

concentration

of

in

cell

lysates

was

measured

by

ELISA

Results

are

means

SE

-LRB-

n

-RRB-

protein

expression

was

examined

at

day

and

day

using

a

specific

ELISA

and

was

detected

at

both

time

points

In

parallel

with

the

mRNA

level

protein

was

substantially

higher

in

preadipocytes

than

in

adipocytes

-LRB-

Fig.

-RRB-

Response

to

treatment

Hypoxic

effects

can

be

mimicked

by

the

divalent

transition-metal

ion

cobalt

In

the

next

experiments

differentiated

human

adipocytes

-LRB-

day

post-induction

-RRB-

were

treated

with

at

concentrations

of

and

μM

for

h.

treatment

resulted

in

a

marked

increase

in

total

protein

the

level

being

fivefold

higher

at

both

doses

compared

to

untreated

cells

-LRB-

Fig.

-RRB-

In

contrast

mRNA

level

was

reduced

three

to

fivefold

by

treatment

-LRB-

Fig.

-RRB-

Fig.

of

at

two

dose

levels

on

protein

concentration

-LRB-

a

-RRB-

and

on

the

mRNA

levels

of

and

adipokines

-LRB-

b

-RRB-

in

human

adipocytes

Differentiated

human

adipocytes

at

day

were

incubated

in

medium

containing

or

μM

for

h.

Total

protein

and

mRNA

level

of

adipokines

were

measured

as

described

in

Fig.

Results

are

means

SE

-LRB-

n

-RRB-

In

a

P

P

compared

with

controls

in

b

P

compared

with

controls

Open

bars

controls

shaded

bars

and

-LRB-

light

and

dark

bars

respectively

-RRB-

To

assess

whether

human

adipocytes

respond

to

the

increase

in

protein

by

increasing

the

expression

of

classical

hypoxia-sensitive

genes

the

mRNA

level

of

the

facilitative

glucose

transporter

was

examined

as

a

reference

this

gene

is

recognised

in

other

cells

to

be

upregulated

under

hypoxic

conditions

Treatment

with

induced

a

sixfold

increase

in

mRNA

level

in

the

adipocytes

-LRB-

Fig.

-RRB-

The

expression

of

a

series

of

inflammation-related

adipokine

genes

was

then

examined

in

the

adipocytes

and

three

distinct

responses

were

observed

No

effect

was

found

on

TNFα

or

adipsin

mRNA

level

at

either

of

the

two

doses

of

There

was

however

a

significant

reduction

in

mRNA

level

for

adiponectin

and

haptoglobin

-LRB-

Fig.

-RRB-

Adiponectin

mRNA

level

was

decreased

three

to

fourfold

by

up

to

fivefold

and

haptoglobin

mRNA

by

less

than

half

with

both

concentrations

In

the

case

of

the

high

dose

of

reduced

mRNA

level

threefold

but

there

was

no

response

with

the

lower

dose

-LRB-

Fig.

-RRB-

In

contrast

treatment

resulted

in

a

marked

increase

in

mRNA

level

for

FIAF

leptin

MIF

and

VEGF

The

increase

was

six

to

sevenfold

for

VEGF

and

FIAF

and

two

to

fourfold

for

MIF

and

leptin

-LRB-

Fig.

-RRB-

Time

course

of

response

to

The

previous

experiment

indicated

that

in

most

cases

there

was

little

difference

in

the

effect

of

or

μM

on

protein

level

and

adipokine

gene

expression

However

with

leptin

MIF

and

in

particular

effects

were

noted

with

the

lower

but

not

the

higher

dose

This

might

suggest

the

possibility

of

additional

or

toxic

responses

to

at

higher

doses

Consequently

in

the

subsequent

time

course

study

a

concentration

of

μM

was

employed

at

day

post-induction

There

was

a

rapid

and

significant

accumulation

of

protein

in

human

adipocytes

after

the

addition

of

The

protein

level

was

increased

by

h

of

treatment

and

peaked

at

h

at

which

point

it

was

increased

before

gradually

falling

-LRB-

Fig.

-RRB-

protein

level

was

also

assessed

by

Western

blotting

and

the

pattern

of

results

was

similar

to

that

obtained

by

ELISA

-LRB-

Fig.

-RRB-

Fig.

course

of

effects

of

-LRB-

μM

-RRB-

on

total

protein

level

-LRB-

a

and

b

-RRB-

and

mRNA

levels

-LRB-

c

-RRB-

in

human

adipocytes

Cells

were

cultured

and

treated

as

in

Fig.

Results

are

means

SE

-LRB-

n

-RRB-

P

P

compared

with

controls

was

again

used

as

a

hypoxia-sensitive

reference

gene

and

the

pattern

of

the

time

course

of

the

changes

in

mRNA

level

was

similar

to

that

of

protein

The

mRNA

level

was

significantly

increased

-LRB-

-RRB-

by

h

peaking

at

h

at

which

point

it

was

higher

gradually

declining

thereafter

-LRB-

Fig.

-RRB-

The

time

course

of

the

effect

of

on

the

expression

of

genes

encoding

adipokines

was

next

examined

A

group

of

adipokines

including

FIAF

leptin

MIF

and

VEGF

showed

marked

increases

in

mRNA

level

in

response

to

some

of

which

were

both

substantial

and

rapid

FIAF

mRNA

level

was

increased

by

h

after

the

addition

of

peaking

at

h

at

which

point

it

had

risen

-LRB-

Fig.

-RRB-

There

was

a

fall

thereafter

but

even

at

h

FIAF

mRNA

was

still

elevated

A

similar

pattern

was

also

evident

with

and

VEGF

but

the

changes

were

not

as

large

-LRB-

Fig.

-RRB-

In

comparison

to

the

above

genes

there

was

a

more

gradual

response

to

with

leptin

and

MIF

the

mRNA

for

each

reaching

a

peak

-LRB-

fourfold

-RRB-

at

h

with

little

change

for

the

rest

of

the

incubation

period

-LRB-

Fig.

-RRB-

Intriguingly

mRNA

level

exhibited

a

substantial

acute

increase

being

elevated

at

h

but

there

was

a

rapid

decline

thereafter

such

that

at

and

h

the

levels

were

similar

to

the

controls

-LRB-

Fig.

-RRB-

Fig.

course

of

effects

of

-LRB-

μM

-RRB-

on

adipokine

mRNA

levels

of

FIAF

leptin

MIF

TNF-α

and

VEGF

-LRB-

a

-RRB-

and

adiponectin

haptoglobin

and

adipsin

-LRB-

b

-RRB-

in

human

adipocytes

Relative

mRNA

levels

Cells

were

cultured

and

treated

as

in

Fig.

Results

are

means

SE

-LRB-

n

-RRB-

P

P

P

compared

with

controls

In

contrast

to

the

genes

described

so

far

no

effect

was

found

on

mRNA

level

throughout

the

incubation

period

in

the

case

of

adipsin

-LRB-

Fig.

-RRB-

while

TNFα

mRNA

was

also

unchanged

except

for

a

transitory

increase

at

h

only

-LRB-

Fig.

-RRB-

There

was

however

a

significant

reduction

in

mRNA

level

for

adiponectin

and

haptoglobin

after

h

of

the

treatment

by

h

adiponectin

and

haptoglobin

mRNA

was

decreased

by

and

threefold

respectively

-LRB-

Fig.

-RRB-

Induction

of

protein

and

gene

expression

by

low

tension

In

the

next

set

of

experiments

the

direct

effects

of

low

tension

were

examined

Human

adipocytes

at

day

post-differentiation

were

exposed

to

normoxic

conditions

-LRB-

-RRB-

or

to

for

up

to

h

Some

cells

were

returned

to

normoxia

for

h

after

h

of

exposure

to

to

examine

the

reversibility

of

responses

Total

protein

level

was

measured

by

both

ELISA

and

Western

blotting

concentration

in

cells

was

rapidly

and

substantially

upregulated

on

exposure

to

being

higher

by

h

than

in

control

cells

cultured

under

normoxia

The

level

fell

markedly

after

h

but

at

h

it

was

still

higher

than

in

control

cells

-LRB-

Fig.

-RRB-

The

transfer

of

adipocytes

back

to

normoxic

conditions

after

h

at

led

to

a

rapid

fall

in

the

level

of

the

protein

at

h

being

threefold

lower

than

in

cells

maintained

in

throughout

-LRB-

the

cells

were

returned

to

normoxia

after

h

rather

than

h

because

the

high

level

of

at

the

earlier

time

point

facilitated

assessment

of

changes

with

normoxia

-RRB-

By

h

after

the

return

to

normoxia

the

level

of

was

similar

to

that

in

the

control

adipocytes

The

pattern

of

level

as

determined

by

ELISA

was

mirrored

by

Western

blot

analysis

-LRB-

Fig.

-RRB-

Fig.

course

of

effects

of

hypoxia

-LRB-

-RRB-

on

total

protein

level

-LRB-

a

b

-RRB-

and

mRNA

-LRB-

c

-RRB-

levels

in

human

adipocytes

Differentiated

adipocytes

at

day

post-induction

were

exposed

to

for

up

to

h

some

cells

were

transferred

back

to

normoxic

conditions

-LRB-

-RRB-

for

or

h

after

h

of

hypoxia

-LRB-

to

and

to

groups

-RRB-

Results

are

means

SE

-LRB-

n

-RRB-

P

P

P

compared

with

controls

mRNA

level

was

markedly

increased

-LRB-

fivefold

-RRB-

after

h

of

exposure

to

-LRB-

Fig.

-RRB-

and

reached

a

peak

at

h

being

higher

than

in

cells

maintained

under

normoxia

After

exposure

to

hypoxia

for

h

the

level

of

mRNA

returned

to

normal

after

h

in

-LRB-

Fig.

-RRB-

Regulation

of

adipokine

gene

expression

by

low

tension

The

effect

of

hypoxia

on

the

expression

of

genes

encoding

key

inflammation-related

adipokines

was

next

examined

The

first

group

of

adipokines

which

encompassed

FIAF

leptin

MIF

and

VEGF

exhibited

rapid

and

marked

increases

in

mRNA

level

in

response

to

hypoxia

as

with

treatment

FIAF

mRNA

level

was

increased

by

h

after

exposure

to

rising

to

by

h

-LRB-

Fig.

-RRB-

FIAF

mRNA

level

was

completely

normalised

by

h

of

reexposure

to

normoxia

after

h

in

A

similar

pattern

was

also

evident

with

five

other

adipokines

The

changes

were

even

more

substantial

in

the

case

of

leptin

and

VEGF

After

h

of

hypoxia

leptin

and

VEGF

mRNA

levels

were

increased

four

to

fivefold

and

rose

such

that

at

h

they

were

and

greater

than

in

controls

respectively

-LRB-

Fig.

-RRB-

Again

after

h

of

reexposure

to

normoxia

after

h

in

the

mRNA

level

of

these

two

adipokines

had

returned

to

normal

Fig.

course

of

effects

of

hypoxia

-LRB-

-RRB-

on

adipokine

gene

expression

in

human

adipocytes.Relative

mRNA

levels

of

FIAF

leptin

MIF

VEGF

-LRB-

a

-RRB-

and

adiponectin

haptoglobin

adipsin

TNF-α

-LRB-

b

-RRB-

Cells

were

cultured

as

in

Fig.

some

cells

were

transferred

back

to

normoxic

conditions

-LRB-

-RRB-

for

or

h

after

h

of

hypoxia

-LRB-

to

and

to

groups

-RRB-

Results

are

means

SE

-LRB-

n

-RRB-

P

P

P

compared

with

controls

MIF

and

mRNA

levels

also

showed

significant

increases

in

response

to

hypoxia

but

these

increases

-LRB-

to

fivefold

at

h

-RRB-

were

less

than

those

exhibited

by

FIAF

leptin

and

VEGF

Returning

the

adipocytes

to

led

to

a

reversal

by

h

of

the

hypoxia-induced

increases

in

MIF

and

mRNA

levels

in

the

case

of

the

mRNA

was

lower

than

in

control

cells

at

h

after

the

transfer

from

hypoxic

to

normoxic

conditions

In

contrast

to

the

genes

described

so

far

there

was

a

significant

reduction

in

adiponectin

and

haptoglobin

mRNA

levels

by

the

end

of

the

exposure

period

the

decrease

being

two

to

threefold

-LRB-

Fig.

-RRB-

This

reduction

was

reversed

after

h

of

reexposure

to

but

surprisingly

was

evident

again

after

h

of

reexposure

Adipsin

mRNA

level

showed

a

significant

but

very

small

increase

at

and

h

-LRB-

and

respectively

-RRB-

There

were

no

significant

changes

in

the

level

of

TNFα

mRNA

during

exposure

to

while

mRNA

was

also

unchanged

after

and

h

of

hypoxia

For

reasons

that

are

not

apparent

there

was

a

significant

reduction

in

mRNA

at

h

and

this

was

sustained

h

after

return

to

normoxia

-LRB-

Fig.

-RRB-

Adipokine

secretion

in

hypoxia

The

secretion

into

the

medium

of

a

selected

group

of

adipokines

was

examined

during

the

exposure

to

hypoxia

using

specific

ELISAs

A

small

but

statistically

significant

reduction

in

adiponectin

secretion

was

observed

after

and

h

incubation

under

-LRB-

Fig.

-RRB-

This

reduction

was

not

evident

after

reexposure

to

for

h

-LRB-

Fig.

-RRB-

Fig.

course

of

effects

of

hypoxia

-LRB-

-RRB-

on

the

release

of

adipokines

from

human

adipocytes

Adiponectin

secretion

-LRB-

a

-RRB-

and

leptin

MIF

and

VEGF

release

-LRB-

b

e

-RRB-

after

exposure

to

hypoxia

Adipokine

concentration

was

measured

by

ELISA

in

the

medium

of

cells

cultured

as

in

Fig.

some

cells

were

transferred

back

to

normoxic

conditions

-LRB-

-RRB-

after

h

of

hypoxia

and

the

adipokine

concentration

then

measured

h

later

-LRB-

to

group

-RRB-

Values

are

means

SE

-LRB-

n

-RRB-

P

P

P

compared

with

controls

In

marked

contrast

to

adiponectin

there

was

a

significant

increase

in

leptin

MIF

and

VEGF

release

after

both

and

h

exposure

to

hypoxia

-LRB-

Fig.

e

-RRB-

The

increases

in

secretion

were

modest

for

MIF

but

substantial

in

the

case

of

both

leptin

and

VEGF

Thus

after

h

of

hypoxia

leptin

release

was

tenfold

higher

while

that

of

VEGF

was

fourfold

greater

Elevated

rates

of

secretion

were

still

evident

after

transfer

of

the

adipocytes

from

hypoxic

to

normoxic

conditions

indicating

a

long-term

carryover

effect

of

low

tension

The

secretion

of

was

not

altered

after

h

of

exposure

to

hypoxia

but

it

was

significantly

increased

at

h

-LRB-

Fig.

-RRB-

Discussion

An

important

recent

development

in

our

understanding

of

obesity

is

the

emergence

of

the

concept

that

it

is

characterised

by

a

state

of

chronic

low-grade

inflammation

-LSB-

-RSB-

White

adipose

tissue

produces

a

number

of

adipokines

linked

to

inflammation

including

adiponectin

TNFα

and

MIF

-LSB-

-RSB-

and

their

synthesis

is

generally

substantially

increased

in

obesity

with

the

exception

of

adiponectin

whose

production

and

circulating

levels

fall

-LSB-

-RSB-

We

have

recently

proposed

that

hypoxia

may

occur

in

adipocytes

distant

from

the

vasculature

as

adipose

tissue

mass

expands

and

that

this

underlies

the

inflammatory

response

exhibited

by

the

tissue

-LSB-

-RSB-

The

central

aim

of

the

present

study

was

to

examine

the

effects

of

hypoxia

on

the

expression

and

secretion

of

key

inflammation-related

adipokines

in

human

adipocytes

The

transcription

factor

is

a

key

signal

in

the

cellular

response

to

hypoxia

the

α-subunit

of

which

is

highly

induced

by

hypoxia

-LSB-

-RSB-

expression

was

evident

in

human

adipocytes

as

noted

previously

in

murine

fat

cells

and

the

level

of

both

the

mRNA

and

the

protein

fell

after

the

induction

of

differentiation

It

has

been

reported

previously

that

mRNA

level

in

cells

peaked

at

h

after

the

addition

of

induction

medium

and

then

rapidly

decreased

-LSB-

-RSB-

Immunoreactive

has

been

reported

in

murine

adipocytes

and

hypoxia

results

in

an

increase

in

the

amount

of

the

protein

in

cultured

fat

cells

-LSB-

-RSB-

We

have

observed

that

the

gene

is

expressed

in

human

mouse

and

rat

WAT

depots

and

expression

occurs

in

both

the

mature

adipocytes

and

in

the

cells

of

the

stromal

vascular

fraction

A

previous

study

has

suggested

that

is

predominantly

expressed

in

the

stromal

vascular

fraction

of

obese

subjects

-LSB-

-RSB-

Hypoxia

stabilises

protein

which

is

otherwise

-LRB-

under

normoxic

conditions

-RRB-

degraded

by

an

ubiquitin-dependent

proteasome

-LSB-

-RSB-

Total

protein

level

in

human

adipocytes

increased

rapidly

and

substantially

under

hypoxic

condition

both

with

and

by

treatment

with

The

elevated

protein

rapidly

fell

on

return

to

and

the

level

was

even

decreased

compared

to

controls

after

h

of

reoxygenation

Thus

the

accumulated

in

human

adipocytes

is

rapidly

degraded

on

returning

to

normoxia

Indeed

it

was

observed

in

another

human

adipocyte

system

-LRB-

SGBS

cells

-RRB-

that

protein

declined

to

control

levels

by

just

min

after

return

to

-LRB-

Wang

unpublished

results

-RRB-

a

known

inducer

of

has

been

widely

employed

as

a

hypoxia

mimic

and

was

used

here

to

examine

the

effects

of

chemically

induced

hypoxia

on

adipokine

gene

expression

Previous

studies

have

shown

that

at

a

global

gene

expression

level

both

and

ambient

hypoxia

regulate

a

similar

group

of

genes

and

the

observed

similarity

in

gene

expression

appears

to

be

dependent

on

functional

-LSB-

-RSB-

stabilises

under

normoxia

via

inhibition

of

the

iron-containing

HIF

prolyl

hydroxylase

enzyme

which

plays

a

critical

role

in

mediating

normal

hypoxic

signalling

by

modifying

and

targeting

it

for

degradation

Our

results

demonstrate

that

rapidly

induces

an

accumulation

of

and

influences

the

expression

of

key

inflammation-related

adipokines

in

human

adipocytes

Other

studies

have

also

found

that

-LRB-

μM

-RRB-

causes

accumulation

in

various

cell

types

including

bone

marrow

stromal

cells

cancer

cells

and

brown

adipocytes

-LSB-

-RSB-

In

contrast

to

protein

levels

mRNA

level

was

inhibited

by

treatment

This

indicates

that

modulates

at

the

posttranslational

rather

than

the

transcriptional

level

There

is

however

evidence

for

hypoxic

induction

of

mRNA

in

some

cells

-LSB-

-RSB-

Under

hypoxia

gene

expression

has

been

shown

to

increase

in

several

cell

types

including

rat

liver

cell

lines

and

fibroblasts

-LSB-

-RSB-

Chinese

hamster

ovary

cells

-LSB-

-RSB-

and

in

human

cancer

cells

-LSB-

-RSB-

Our

data

demonstrate

that

expression

is

also

substantially

upregulated

by

hypoxic

conditions

in

human

adipocytes

Measurement

by

Western

blotting

-LRB-

using

a

specific

antibody

-RRB-

of

in

total

tissue

lysates

of

adipocytes

exposed

to

demonstrated

that

the

level

of

the

protein

was

also

increased

by

hypoxia

at

h

protein

was

eightfold

higher

than

in

the

normoxic

controls

-LRB-

Wood

unpublished

results

-RRB-

This

phenomenon

presumably

reflects

an

adaptation

of

glucose

metabolism

to

hypoxic

conditions

with

a

marked

stimulation

of

glycolysis

in

the

face

of

a

fall

in

aerobic

metabolism

During

the

adaptive

response

to

hypoxia

the

expression

of

several

genes

encoding

glycolytic

enzymes

is

recognised

to

increase

-LSB-

-RSB-

To

sustain

higher

levels

of

glycolysis

there

is

a

need

for

an

increase

in

glucose

uptake

and

this

would

be

aided

by

an

increase

in

the

level

of

expression

in

hypoxia

is

a

key

facilitative

glucose

transporter

in

white

adipocytes

but

other

transporter

isoforms

such

as

and

are

also

expressed

in

the

cells

-LSB-

-RSB-

The

main

focus

of

the

present

study

was

the

effects

of

hypoxia

on

the

expression

and

secretion

of

adipokines

and

qualitatively

similar

results

were

obtained

over

the

same

time

scale

with

both

and

treatment

Given

the

mechanism

of

action

of

this

is

consistent

with

the

effects

of

hypoxia

on

adipokine

production

being

mediated

by

Of

the

adipokines

studied

the

expression

of

TNFα

and

adipsin

were

not

altered

and

these

genes

would

seem

not

to

be

hypoxia-sensitive

However

the

expression

of

both

adiponectin

and

haptoglobin

-LRB-

an

acute

phase

protein

-RRB-

in

human

adipocytes

was

reduced

by

hypoxic

conditions

and

in

the

case

of

adiponectin

this

was

also

evident

at

the

level

of

the

secretion

of

the

protein

into

the

medium

The

reduction

albeit

modest

in

adiponectin

production

in

human

adipocytes

by

hypoxia

is

consistent

with

a

recent

report

in

murine

adipocytes

-LSB-

-RSB-

There

was

a

discrepancy

in

the

present

study

between

the

changes

in

the

scale

of

alterations

in

adiponectin

mRNA

level

and

the

amount

of

adiponectin

secreted

into

the

medium

This

may

however

reflect

the

lag

between

changes

in

mRNA

and

alterations

in

secreted

protein

In

marked

contrast

to

adiponectin

hypoxia

stimulated

the

expression

of

the

genes

encoding

FIAF

leptin

MIF

and

VEGF

VEGF

is

involved

in

the

development

of

the

vascular

system

promoting

angiogenesis

and

is

well

known

to

be

activated

by

hypoxia

-LSB-

-RSB-

In

the

present

study

an

increase

in

VEGF

protein

secretion

as

well

as

mRNA

level

was

observed

in

human

adipocytes

with

hypoxia

This

is

in

agreement

with

studies

on

rat

omental

adipose

tissue

and

on

murine

adipocytes

-LSB-

-RSB-

Leptin

is

the

most

extensively

studied

adipokine

and

adipose

tissue

is

quantitatively

the

main

leptin-producing

organ

Activation

of

leptin

gene

expression

by

hypoxia

resulted

in

a

substantial

increase

in

leptin

release

by

human

adipocytes

This

would

be

expected

to

reduce

food

intake

and

stimulate

energy

expenditure

and

as

such

hypoxia

could

provide

a

mechanistic

basis

for

the

increase

in

leptin

as

adipose

tissue

mass

expands

in

obesity

Leptin

has

been

reported

to

be

an

angiogenic

factor

-LSB-

-RSB-

and

the

physiological

significance

of

a

hypoxia-mediated

induction

of

leptin

secretion

by

adipocytes

may

relate

to

a

local

effect

to

stimulate

the

development

of

new

blood

vessels

during

adipose

tissue

expansion

Therefore

increased

leptin

production

together

with

that

of

VEGF

under

hypoxic

conditions

could

also

reflect

an

adaptive

process

to

promote

angiogenesis

allowing

appropriate

oxygenation

of

adipose

tissue

It

has

been

previously

established

that

leptin

is

a

hypoxia-inducible

hormone

and

its

production

is

increased

by

hypoxia

in

tissues

such

as

the

placenta

cardiac

myocytes

and

cancer

cells

-LSB-

-RSB-

as

well

as

in

murine

adipocytes

-LSB-

-RSB-

FIAF

-LRB-

also

known

as

angiopoietin-like

protein

-RRB-

is

expressed

particularly

in

adipose

tissue

and

the

liver

and

has

been

suggested

to

play

a

role

in

adipose

differentiation

systemic

lipid

metabolism

and

energy

homeostasis

-LSB-

-RSB-

In

parallel

with

the

activation

of

leptin

and

VEGF

FIAF

expression

was

highly

activated

by

hypoxia

in

human

adipocytes

FIAF

expression

has

also

been

shown

to

be

hypoxia-sensitive

in

cardiomyocytes

through

a

pathway

-LSB-

-RSB-

The

major

upregulation

of

FIAF

by

hypoxia

in

adipocytes

may

again

relate

to

angiogenesis

this

being

one

of

the

functions

suggested

for

this

protein

-LSB-

-RSB-

However

a

major

effect

of

FIAF

is

in

the

clearance

of

plasma

triglycerides

through

an

inhibition

of

lipoprotein

lipase

and

it

also

appears

to

stimulate

lipolysis

-LSB-

-RSB-

Thus

the

hypoxia-induced

stimulation

of

FIAF

production

may

alter

lipid

utilisation

The

present

study

shows

that

the

expression

and

release

of

MIF

a

potent

macrophage

migration

inhibitory

factor

which

has

recently

been

shown

to

be

secreted

from

human

adipocytes

-LSB-

-RSB-

was

also

stimulated

by

hypoxia

MIF

has

been

described

as

a

pro-inflammatory

agent

and

elevated

expression

has

been

seen

in

many

inflammatory

pathologies

Although

MIF

may

play

a

role

in

the

normal

adaptive

response

to

hypoxia

it

is

likely

that

exaggerated

expression

of

MIF

may

contribute

to

the

inflammatory

state

It

has

also

been

proposed

that

MIF

may

play

an

important

role

in

oncogenic

transformation

and

tumour

survival

and

recent

studies

have

implicated

MIF

in

the

regulation

of

VEGF

expression

-LSB-

-RSB-

In

this

context

during

hypoxia

elevated

MIF

expression

may

play

an

important

role

in

enhancing

angiogenesis

as

well

as

macrophage

recruitment

In

contrast

to

MIF

no

consistent

pattern

was

observed

with

an

important

factor

in

macrophage

recruitment

the

acute

but

transitory

increase

in

mRNA

observed

with

may

reflect

an

effect

of

the

metal

unrelated

to

hypoxia

The

expression

and

release

of

the

classic

inflammatory

factor

was

significantly

upregulated

by

hypoxia

Thus

hypoxia

can

directly

affect

key

components

of

the

inflammatory

cascade

within

adipose

tissue

The

expression

of

an

acute

phase

protein

involved

in

fibrinolysis

was

modestly

increased

in

human

adipocytes

by

hypoxia

consistent

with

the

effects

observed

in

adipocytes

-LSB-

-RSB-

Induction

of

expression

by

hypoxia

has

also

been

reported

in

cells

-LSB-

-RSB-

and

in

vascular

smooth

muscle

cells

-LSB-

-RSB-

Overall

the

present

results

indicate

that

a

number

of

genes

are

hypoxia-sensitive

in

human

adipocytes

and

suggest

that

is

involved

in

the

transmission

of

the

response

to

low

tension

in

these

as

in

other

cell

types

The

hypoxia-sensitive

genes

in

adipocytes

include

those

involved

in

basic

metabolic

processes

such

as

glucose

transport

as

well

as

those

linked

to

energy

balance

inflammation

and

angiogenesis

The

stimulation

of

leptin

MIF

and

VEGF

expression

together

with

the

downregulation

of

adiponectin

synthesis

is

consistent

with

our

recent

proposal

that

hypoxia

may

underlie

the

inflammatory

response

in

adipose

tissue

in

obesity

and

play

a

causal

role

in

the

development

of

obesity-associated

diseases

-LSB-

-RSB-

There

was

however

no

effect

of

hypoxia

on

the

expression

of

TNFα

a

key

pro-inflammatory

cytokine

The

hypoxia-induced

changes

in

and

adiponectin

with

its

anti-inflammatory

action

in

particular

might

be

expected

to

impact

on

insulin

sensitivity

O O O O O O O O B O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

adipokines
adipose tissue
hypoxia
inflammation
obesity
metabolic syndrome


The CD38/CD157 mammalian gene family : An evolutionary paradigm for other leukocyte surface enzymes Human CD38 is the mammalian prototype of a family of phylogenetically conserved proteins which share structural similarities and enzymatic activities involved in the production of an intracellular second messenger with calcium mobilizing effects . Engagement of CD38 by agonistic monoclonal antibodies and the CD31 ligand initiates a cytoplasmic signaling cascade involving tyrosine phosphorylation of the proto-oncogene c-cbl and of the extracellular regulated kinase 1 of 2 complex . Further requirements for signal transduction include a privileged localization within the cholesterol-rich areas of the plasma membrane and physical association with specialized surface receptors . CD38-mediated signals are crucial in heterotypic cell adhesion and migration as well as in the activation of proliferation/survival programs by normal and neoplastic cells . Here we review the most recent literature on this complex topic and attempt to formulate a single model reconciling the enzymatic and receptorial activities of CD38 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

calcium mobilization
cell surface enzymes
cell surface receptors
intracellular signaling