Absence

of

an

adipogenic

effect

of

rosiglitazone

on

mature

adipocytes

increase

of

lipid

catabolism

and

reduction

of

adipokine

expression

Aims/hypothesis

The

thiazolidinedione

-LRB-

TZD

-RRB-

rosiglitazone

is

a

peroxisome

proliferator-activated

receptor-γ

agonist

that

induces

adipocyte

differentiation

and

hence

lipid

accumulation

This

is

in

apparent

contrast

to

the

long-term

glucose-lowering

insulin-sensitising

effect

of

rosiglitazone

We

tested

whether

the

action

of

rosiglitazone

involves

specific

effects

on

mature

adipocytes

which

are

different

from

those

on

preadipocytes

Introduction

Rosiglitazone

-LRB-

-RRB-

a

thiazolidinedione

-LRB-

TZD

-RRB-

drug

is

used

in

the

treatment

of

type

diabetes

mellitus

-LSB-

-RSB-

It

has

been

proposed

that

the

glucose-lowering

action

of

TZDs

is

mainly

mediated

by

activating

peroxisome

proliferator-activated

receptor

-LRB-

PPAR

-RRB-

γ

-LRB-

PPARG

-RRB-

-LSB-

-RSB-

PPARG

is

most

abundantly

produced

in

adipose

tissue

suggesting

that

this

is

the

primary

site

of

action

of

TZDs

-LSB-

-RSB-

Proposed

mechanisms

underlying

the

adipocyte-mediated

glucose-lowering

action

of

TZDs

are

the

trapping

of

fatty

acids

inside

adipocytes

and

away

from

muscle

the

so-called

fatty

acid

steal

hypothesis

and

the

altering

adipokine

release

-LSB-

-RSB-

PPARG

is

a

critical

transcription

factor

in

adipogenesis

-LSB-

-RSB-

Its

production

increases

strongly

during

adipocyte

differentiation

-LSB-

-RSB-

By

activating

PPARG

rosiglitazone

promotes

adipocyte

differentiation

in

vitro

-LSB-

-RSB-

An

increase

in

the

number

of

small

adipocytes

and

fat

mass

by

TZDs

in

both

animal

models

and

human

subjects

suggests

that

adipocyte

differentiation

also

occurs

in

vivo

-LSB-

-RSB-

Compared

with

preadipocytes

adipocytes

have

a

much

higher

capacity

for

fatty

acid

uptake

and

lipid

storage

and

are

potentially

more

insulin-sensitive

due

to

higher

numbers

of

glucose

transporters

and

insulin

receptors

-LSB-

-RSB-

By

increasing

the

number

of

adipocytes

TZDs

can

lower

circulating

NEFA

and

improve

lipid

exposure

of

peripheral

tissues

thus

improving

whole-body

insulin

sensitivity

-LSB-

-RSB-

This

hypothesis

is

clearly

attractive

but

other

effects

of

TZDs

may

occur

It

has

been

pointed

out

that

the

increased

number

of

small

adipocytes

caused

by

TZD

treatment

is

not

only

a

result

of

the

appearance

of

new

adipocytes

but

also

due

to

the

shrinkage

and/or

disappearance

of

existing

mature

adipocytes

-LSB-

-RSB-

TZD-induced

body

weight

gain

in

vivo

is

used

as

a

marker

of

increased

adiposity

However

there

is

evidence

that

this

may

also

be

due

to

fluid

retention

-LSB-

-RSB-

In

fact

the

fat

mass

may

be

unaffected

or

even

decreased

In

ob/ob

mouse

whole

body

weight

was

increased

but

the

fat

pad

weight

was

not

changed

after

rosiglitazone

treatment

-LSB-

-RSB-

In

type

diabetes

patients

the

visceral

fat

area

was

found

to

be

decreased

-LSB-

-RSB-

Fatty

acid

steal

therefore

is

not

just

a

matter

of

increased

lipid

storage

The

maturation

of

adipocytes

is

positively

correlated

with

the

production

of

adipokines

Several

of

these

profoundly

influence

insulin

sensitivity

and

glucose

metabolism

e.g.

leptin

adiponectin

and

resistin

-LSB-

-RSB-

However

it

has

been

observed

that

TZDs

decrease

the

circulating

level

of

individual

adipokines

such

as

leptin

-LSB-

-RSB-

resistin

-LSB-

-RSB-

and

plasminogen

activator

-LSB-

-RSB-

and

also

of

cytokines

-LSB-

-RSB-

A

decreased

production

of

these

adipokines

by

TZDs

seems

to

conflict

with

the

adipogenic

properties

of

these

drugs

The

above

discrepancy

could

be

due

to

different

effects

on

preadipocytes

and

mature

adipocytes

in

adipose

tissue

It

is

clear

that

TZDs

stimulate

preadipocytes

into

the

process

of

differentiation

However

their

effect

on

mature

adipocytes

is

less

clear

In

vivo

studies

do

not

allow

us

to

distinguish

between

the

effects

on

these

two

cell

types

Therefore

to

study

the

effects

of

TZDs

on

mature

adipocytes

we

used

in

vitro

differentiated

mature

adipocytes

In

addition

to

the

assessment

of

lipid

accumulation

and

adipokine

secretion

we

used

transcriptomics

to

obtain

insight

into

the

effects

of

rosiglitazone

Although

high

insulin

and

high

glucose

levels

mimic

the

situation

in

vivo

when

rosiglitazone

is

administered

we

also

included

low

insulin

and

low

glucose

levels

for

comparison

Materials

and

methods

Cell

culture

and

sample

preparation

Murine

fibroblasts

-LRB-

American

Type

Culture

Collection

Manassas

VA

US

-RRB-

were

cultured

and

differentiated

to

adipocytes

in

vitro

as

described

-LSB-

-RSB-

On

day

differentiated

cells

grown

in

DMEM/F

-LRB-

-RRB-

supplemented

with

fetal

calf

serum

-LRB-

Perbio

Science

Erembodegem

Belgium

-RRB-

were

treated

with

μmol/l

rosiglitazone

maleate

-LRB-

GlaxoSmithKline

Worthing

UK

-RRB-

with

low

-LRB-

mmol/l

-RRB-

glucose

and

-LRB-

fetal

calf

serum

level

-RRB-

insulin

-LRB-

Rosi

-RRB-

or

with

high

-LRB-

mmol/l

-RRB-

glucose

and

μmol/l

insulin

-LRB-

RosiIG

-RRB-

In

parallel

control

cells

were

cultured

in

the

same

medium

with

low

-LRB-

basal

-RRB-

or

high

glucose

and

insulin

-LRB-

IG

-RRB-

Each

condition

was

created

in

quadruplicate

On

day

two

replications

were

collected

separately

for

total

RNA

isolation

using

Trizol

as

described

-LSB-

-RSB-

Cells

of

the

other

two

replications

were

washed

and

incubated

with

the

corresponding

serum-free

medium

supplemented

with

mg/l

transferrin

and

nmol/l

sodium

selenite

for

h

The

number

of

cells

was

counted

and

culture

medium

proteins

were

collected

as

described

-LSB-

-RSB-

The

duplicated

protein

samples

were

pooled

for

later

analysis

Three

independent

experiments

were

performed

Oil

red

O

staining

An

optimised

Oil

Red

O

staining

method

-LSB-

-RSB-

was

adapted

for

adipocytes

cultured

in

a

six-well

plate

We

added

an

additional

quick

wash

with

ethanol

after

fixing

and

washing

with

water

After

staining

and

washing

the

neutral

lipid-bound

pigment

was

dissolved

in

DMSO

and

absorbance

at

nm

was

measured

The

staining

for

preadipocytes

was

used

as

background

to

correct

for

unspecific

binding

The

obtained

absorbance

values

were

further

corrected

for

cell

numbers

measured

in

replicated

wells

Five

independent

experiments

were

performed

in

duplicate

but

in

the

first

two

experiments

Basal

and

Rosi

conditions

were

not

measured

Glycerol

assay

The

culture

media

of

cells

that

had

been

treated

for

h

were

collected

per

condition

frozen

in

liquid

then

stored

at

°C

until

analysis

The

concentration

of

glycerol

in

the

medium

was

measured

by

a

quantitative

enzymatic

assay

kit

-LRB-

Sigma

St

Louis

MO

USA

-RRB-

according

to

the

manufacturer

's

instructions

The

concentrations

were

corrected

for

cell

numbers

measured

in

replicated

wells

in

parallel

Microarray

The

mouse

oligo

set

-LRB-

MWG

Ebersberg

Germany

-RRB-

and

additional

oligonucleotides

of

genes

encoding

secretory

proteins

and

proteins

involved

in

energy

metabolism

were

printed

in-house

and

further

annotated

as

described

-LSB-

-RSB-

Five

total

RNA

samples

of

three

independent

experiments

per

condition

were

used

for

hybridisation

against

reference

RNA

also

as

described

-LSB-

-RSB-

Microarray

data

analysis

The

data

were

analysed

as

described

-LSB-

-RSB-

Briefly

spots

with

an

average

intensity

over

all

arrays

of

lower

than

twofold

above

average

background

were

removed

from

further

analysis

then

the

intensities

of

the

remaining

spots

were

normalised

against

reference

Fold-change

calculations

Student

's

t

tests

and

correlation

test

-LRB-

Pearson

-RRB-

were

performed

in

Excel

-LRB-

Microsoft

-RRB-

Fold-change

equals

ratio

in

the

case

of

increase

and

equals

in

the

case

of

decrease

Cut-off

criteria

for

differential

expression

was

set

at

fold-change

and

p

For

genes

with

replicated

spots

the

average

value

of

individual

spots

was

used.Data

were

further

analysed

using

Ingenuity

Pathways

Analysis

-LRB-

Ingenuity

Systems

-RRB-

The

program

recognised

of

spot

entries

and

thus

genes

Differentially

expressed

genes

that

associated

with

a

canonical

pathway

in

the

Ingenuity

Pathways

Knowledge

Base

were

considered

for

evaluation

The

significance

of

the

association

between

the

data

set

and

the

canonical

pathway

was

measured

in

two

ways

-LRB-

-RRB-

a

ratio

of

the

number

of

genes

from

the

data

set

that

map

to

the

pathway

divided

by

the

total

number

of

genes

that

map

to

the

canonical

pathway

is

displayed

-LRB-

-RRB-

Fischer

's

exact

test

was

used

to

calculate

a

p

value

determining

the

probability

that

the

association

between

the

genes

in

the

dataset

and

the

canonical

pathway

is

explained

by

chance

alone

A

pathway

with

a

significance

was

taken

as

significantly

regulated.Quantitative

real-time

RT-PCR

All

six

total

RNA

samples

per

treatment

were

used

for

quantitative

real-time

RT-PCR

-LRB-

Q-PCR

-RRB-

of

genes

-LRB-

primer

information

in

Electronic

supplementary

material

-LSB-

ESM

-RSB-

Table

-RRB-

Q-PCR

was

performed

as

described

-LSB-

-RSB-

Ribosomal

protein

-LRB-

-RRB-

mRNA

was

used

as

reference

One

and

two-dimensional

gel

electrophoresis

Protein

samples

were

analysed

by

one-dimensional

-LRB-

-RRB-

and

two-dimensional

-LRB-

-RRB-

gel

electrophoresis

corrected

for

cell

numbers

as

described

-LSB-

-RSB-

Brefeldin-A-treated

adipocyte

medium

protein

sample

-LSB-

-RSB-

was

loaded

on

the

same

gel

to

verify

protein

secretion

Differentially

expressed

secreted

protein

bands

were

excised

and

identified

by

matrix-assisted

laser

desorption

ionisation-time

of

flight

mass

spectrometry

-LSB-

-RSB-

Western

blotting

The

secretion

level

of

adiponectin

was

analysed

by

western

blotting

as

described

-LSB-

-RSB-

Results

Effect

of

rosiglitazone

on

the

lipid

content

of

mature

adipocytes

To

assess

the

effect

of

rosiglitazone

on

lipid

accumulation

in

mature

adipocytes

we

measured

the

lipid

content

of

differentiated

adipocytes

Visual

inspection

by

microscopy

showed

no

change

in

morphology

and

fat

cell

percentage

in

the

population

of

adipocytes

after

days

of

treatment

with

RosiIG

or

Rosi

-LRB-

μmol/l

rosiglitazone

at

either

high

or

low

insulin/glucose

levels

respectively

-RRB-

as

compared

with

untreated

adipocytes

Nevertheless

the

lipid

content

as

detected

by

Oil

Red

O

staining

was

lower

-LRB-

p

n

-RRB-

in

RosiIG-treated

cells

while

no

difference

was

observed

in

Rosi-treated

cells

-LRB-

Fig.

-RRB-

Fig.

Red

O

staining

-LRB-

ORO

-RRB-

for

lipid

content

in

adipocytes

Data

are

expressed

as

mean

SD

n

for

high

glucose

and

insulin

-LRB-

control

condition

-RRB-

-LRB-

IG

-RRB-

and

rosiglitazone

with

high

insulin

and

glucose

condition

-LRB-

RosiIG

-RRB-

n

for

basal

and

rosiglitazone

with

low

insulin

and

glucose

condition

-LRB-

Rosi

-RRB-

Blank

bars

absorbance

relative

value

filled

bars

absorbance

relative

value

corrected

for

cell

numbers

p

compared

with

IG

condition

before

cell

number

correction

-LRB-

t

test

-RRB-

p

compared

with

IG

condition

after

cell

number

correction

-LRB-

t

test

-RRB-

Effect

of

rosiglitazone

on

gene

expression

To

reveal

the

possible

mechanisms

for

the

decreased

lipid

content

of

rosiglitazone-treated

adipocytes

we

used

DNA

microarray

to

profile

the

gene

expression

The

complete

analysis

set

was

imported

into

Ingenuity

Pathway

Analysis

for

canonical

pathway

analysis

The

significantly

changed

pathways

were

involved

in

metabolism

but

not

in

signalling

such

as

PPAR

signalling

or

insulin

receptor

signalling

-LRB-

Fig.

-RRB-

The

expression

of

Pparg

itself

was

downregulated

and

-LRB-

p

-RRB-

by

Rosi

and

RosiIG

treatment

respectively

Notably

pathways

related

to

energy

metabolism

were

among

those

that

were

most

changed

Taking

the

high

redundancy

among

these

pathways

in

the

Ingenuity

database

into

account

we

checked

the

individual

pathways

for

redundancy

This

showed

that

oxidative

phosphorylation

fatty

acid

metabolism

glycolysis

and

gluconeogenesis

the

pentose

phosphate

pathway

the

citric

acid

-LRB-

TCA

-RRB-

cycle

glycerolipid

metabolism

glutathione

metabolism

steroid

-LRB-

androgen

oestrogen

and

steroids

-RRB-

metabolism

and

prostaglandin

metabolism

are

independent

pathways

while

for

the

others

it

can

not

be

ruled

out

that

they

were

identified

as

significant

just

because

they

contain

genes

overlapping

with

other

pathways

Fig.

canonical

pathways

by

rosiglitazone

Microarray

data

analysed

by

Ingenuity

Pathway

Analysis

Blank

bars

with

low

insulin/glucose

filled

bars

with

high

insulin/glucose

Bold

line

thresholdFor

these

independently

regulated

pathways

we

determined

the

direction

of

change

by

examining

the

individual

genes

in

the

pathways

The

regulation

of

gene

expression

of

rate-limiting

or

otherwise

representative

enzymes

is

presented

in

Table

High

insulin/glucose

attenuated

the

effect

of

rosiglitazone

on

some

genes

of

complex

I

and

II

of

the

electron

transport

chain

However

high

insulin/glucose

treatment

itself

did

not

induce

a

change

in

the

expression

of

these

genes

from

the

basal

state

In

general

rosiglitazone

produced

dominant

effects

as

indicated

by

the

Pearson

product

moment

correlation

coefficient

of

for

the

fold

changes

of

the

complete

analysis

set

of

Rosi/Basal

and

RosiIG/IG

The

changes

in

the

pathways

indicate

that

rosiglitazone

enhanced

both

energy

metabolism

at

the

TCA

cycle

and

oxidative

phosphorylation

The

generated

energy

may

be

dispensed

with

the

help

of

upregulated

uncoupling

protein

-LRB-

-RRB-

Enhanced

energy

metabolism

is

mainly

supported

by

upregulation

of

the

pathways

involved

in

uptake

and

activation

of

fatty

acids

and

in

beta-oxidation

in

both

mitochondria

and

peroxisomes

In

contrast

de

novo

fatty

acid

synthesis

was

downregulated

as

indicated

by

fatty

acid

synthase

-LRB-

Fasn

-RRB-

and

stearoyl-coenzyme

A

desaturase

-LRB-

-RRB-

The

downregulation

of

triacylglycerol

synthesis

enzymes

and

upregulation

of

adipocyte

lipase

-LRB-

also

known

as

patatin-like

phospholipase

domain

containing

-RRB-

together

imply

a

decreased

lipogenesis

in

mature

adipocytes

-LRB-

Table

-RRB-

Table

enzymes

of

metabolism

pathways

regulated

by

rosiglitazonePathwayChangeGene

symbolGene

IDNameRosi/BasalRosiIG/IGFold-changep

valueFold

changep

valueTCA

dehydrogenase

-LRB-

NAD

-RRB-

Oxidative

dehydrogenase

-LRB-

ubiquinone

-RRB-

alpha

subcomplex

dehydrogenase

complex

subunit

c

reductase

core

protein

c

oxidase

subunit

VIIa

polypeptide

synthase

H

transporting

mitochondrial

complex

subunit

Fatty

acid

uptake

and

antigen

fatty

acid

acid

bind

protein

Fatty

acid

synthetase

long-chain

family

member

Fatty

acid

beta

carrier

family

-LRB-

mitochondrial

carnitine/acylcarnitine

translocase

-RRB-

member

A

oxidase

A

acyltransferase

-LRB-

peroxisomal

-RRB-

A

acyltransferase

-LRB-

mitochondrial

-RRB-

Fatty

acid

acid

synthase

desaturase

Triacylglycerol

acid

phosphatase

type

O-acyltransferase

Lipid

droplet

differentiation

related

caveolae

protein

Glycerolipid

phospholipase

domain

containing

Glycerol

dehydrogenase

-LRB-

soluble

-RRB-

Energy

protein

-LRB-

mitochondrial

proton

carrier

-RRB-

Glutathione

glutathione

S-transferase

S-transferase

S-transferase

S-transferase

theta

transferase

zeta

Prostaglandin

reductase

E

synthase

-LRB-

prostacyclin

-RRB-

synthase

Steroids

dehydrogenase

-LRB-

P

-RRB-

dependent

steroid

dehydrogenase

Glycogen

Pyruvate

dehydrogenase

alpha

dehydrogenase

-LRB-

lipoamide

-RRB-

Pentose

dehydrogenase

-LRB-

glucose

-RRB-

Leu

coenzyme

A

dehydrogenase

A

carboxylase

-LRB-

alpha

-RRB-

Mean

value

of

microarray

data

-LRB-

n

-RRB-

Rosi/basal

the

effect

with

low

insulin/glucose

RosiIG/IG

the

effect

with

high

insulin/glucoseAccompanying

fatty

acid

catabolism

glycerol

activation

by

glycerol

kinase

and

oxidation

by

glycerol

phosphate

dehydrogenase

to

dihydroxyacetone

phosphate

were

upregulated

feeding

glycolysis

and

gluconeogenesis

The

enzymes

catalysing

glycolysis/gluconeogenesis

were

all

upregulated

possibly

in

favour

of

gluconeogenesis

In

addition

the

upregulation

of

glycogenin

a

gene

priming

glycogen

synthesis

implies

an

increase

of

glycogen

synthesis.Most

genes

in

the

amino

acid

metabolism

pathways

that

were

regulated

also

participate

in

fatty

acid

metabolism

or

in

glycolysis

Therefore

we

did

not

take

amino

acid

metabolism

pathways

as

independent

rosiglitazone-regulated

pathways

However

two

key

enzymes

that

catalyse

the

catabolism

of

leucine

-LRB-

Table

-RRB-

were

downregulated

This

implies

that

rosiglitazone

saves

amino

acids

from

being

used

as

fuel

in

energy

metabolism.Glutathione

metabolism

was

significantly

changed

by

rosiglitazone

treatment

Two

glutathione

S-transferases

that

detoxify

lipid

peroxides

microsomal

glutathione

S-transferase

and

glutathione

S-transferase

were

upregulated

while

other

glutathione

S-transferases

were

downregulated

by

rosiglitazone

This

is

in

line

with

the

upregulation

of

fatty

acid

oxidation

by

rosiglitazone.Microsomal

glutathione

S-transferase

is

involved

not

only

in

glutathione

metabolism

but

also

in

eicosanoid

metabolism

which

is

also

regulated

by

rosiglitazone

The

expression

of

genes

encoding

enzymes

involved

in

prostaglandin

metabolism

implies

that

the

conversion

from

prostaglandin

to

and

may

be

enhanced

while

conversion

to

may

be

reduced.Cholesterol

biosynthesis

and

steroid

hormone

metabolism

were

also

affected

by

rosiglitazone

Expression

of

some

steroid

dehydrogenases

were

upregulated

while

hydroxysteroid

dehydrogenase

was

downregulated

-LRB-

Table

-RRB-

The

latter

has

been

reported

as

one

of

the

beneficial

effects

of

TZDs

leading

to

a

decrease

in

the

stress

hormone

cortisol

-LSB-

-RSB-

Effect

of

rosiglitazone

on

the

expression

of

genes

encoding

adipocyte-secreted

proteins

To

assess

whether

protein

secretion

by

mature

adipocytes

is

altered

by

rosiglitazone

we

checked

genes

encoding

adipocyte-secreted

proteins

The

list

was

based

on

literature

-LSB-

-RSB-

and

our

previous

work

-LSB-

-RSB-

In

total

genes

were

present

on

the

array

and

leptin

showed

very

low

signals

-LRB-

less

than

twofold

of

background

-RRB-

and

were

excluded

from

the

analysis

Among

the

remaining

genes

only

apolipoprotein

E

-LRB-

Apoe

-RRB-

was

upregulated

by

Rosi

treatment

and

more

than

of

the

genes

were

significantly

downregulated

with

both

Rosi

and

RosiIG

treatment

-LRB-

Table

-RRB-

Table

of

rosiglitazone

on

the

expression

of

genes

encoding

adipocyte-secreted

proteinsFunctionGene

symbolGene

IDNameRosi/BasalRosiIG/IGFold-changep

valueFold-changep

valueLipid

factor

D

-LRB-

adipsin

-RRB-

component

Insulin

growth

factor

colony-enhancing

factor

gene

growth

factor

beta

microglobulin

-LRB-

C-C

motif

-RRB-

ligand

-LRB-

previously

known

as

monocyte

chemoattractant

-RRB-

amyloid

A

-LRB-

C-X-C

motif

-RRB-

ligand

receptor

alpha

growth

factor

beta

growth

factor

beta

segment

Chr

Wayne

State

University

expressed

-LRB-

previously

known

as

-RRB-

galactose

binding

soluble

migration

inhibitory

Vascular

-LRB-

or

cysteine

-RRB-

peptidase

inhibitor

clade

F

member

-LRB-

previously

known

as

pigment

epithelium

derived

factor

-RRB-

endothelial

growth

factor

A

-LRB-

or

cysteine

-RRB-

peptidase

inhibitor

clade

E

member

-LRB-

previously

known

as

plasminogen

activator

-RRB-

dismutase

extracellular

Extracellular

matrix

acidic

cysteine

rich

glycoprotein

type

V

alpha

type

V

alpha

type

I

alpha

type

I

alpha

type

III

alpha

type

IV

alpha

type

VI

alpha

type

VI

alpha

type

V

alpha

type

IV

alpha

type

IV

alpha

type

VI

alpha

Extracellular

matrix

metalloproteinase

oxidase

inhibitor

of

metalloproteinase

metalloproteinase

inhibitor

of

metalloproteinase

C-endopeptidase

enhancer

protein

metalloproteinase

metalloproteinase

inhibitor

of

metalloproteinase

isomerase

C

Mean

value

of

microarray

data

-LRB-

n

-RRB-

Rosi/basal

the

effect

with

low

insulin/glucose

RosiIG/IG

the

effect

with

high

insulin/glucoseInterestingly

the

general

downregulation

of

adipokines

was

also

observed

for

other

genes

encoding

secreted

proteins

We

used

Gene

Ontology

to

cluster

the

expressed

genes

on

the

microarray

into

secreted

protein

encoding

genes

and

intracellular/unknown

location

protein-encoding

genes

-LRB-

additional

gene

expression

information

is

available

at

-RRB-

In

the

first

cluster

more

genes

were

significantly

downregulated

-LRB-

Fig.

and

c

-RRB-

Of

genes

and

were

significantly

downregulated

while

and

were

upregulated

by

Rosi

and

RosiIG

respectively

Of

the

upregulated

genes

most

encode

multi-location

proteins

which

can

also

reside

in

the

nucleus

mitochondria

and/or

membranes

If

these

genes

are

omitted

from

the

analysis

only

seven

genes

were

upregulated

-LRB-

-RRB-

In

the

cellular

protein

cluster

by

contrast

the

majority

of

the

regulated

genes

were

upregulated

-LRB-

Fig.

and

d

-RRB-

Of

genes

and

were

significantly

upregulated

while

and

were

downregulated

by

Rosi

and

RosiIG

treatment

respectively

Fig.

pattern

by

rosiglitazone

of

genes

encoding

extracellular

proteins

and

of

genes

encoding

intracellular

proteins

or

proteins

of

unknown

location

a

c

extracellular

pattern

b

d

intracellular

pattern

a

b

rosiglitazone

with

low

insulin/glucose

c

d

rosiglitazone

with

high

insulin/glucoseTo

complement

array

data

we

used

and

gel

electrophoresis

and

western

blotting

to

examine

the

effect

of

rosiglitazone

on

secretion

With

regard

to

complement

a

reduction

of

secretion

by

Rosi

or

RosiIG

treatment

was

observed

on

the

gel

-LRB-

Fig.

-RRB-

This

is

less

than

the

change

observed

at

the

gene

expression

level

Nine

spots

on

the

gel

were

identified

as

adipsin

We

assessed

the

changes

for

individual

spots

as

well

as

for

all

spots

in

total

In

both

cases

the

decrease

effected

by

rosiglitazone

at

the

gene

expression

level

was

similar

to

the

decrease

found

at

the

secreted

protein

level

with

RosiIG

treatment

However

this

decrease

at

the

protein

level

was

less

prominent

with

Rosi

treatment

-LRB-

Fig.

-RRB-

The

secretion

of

adiponectin

was

also

analysed

by

western

blotting

A

slight

increase

with

rosiglitazone

was

detected

-LRB-

Fig.

-RRB-

although

there

was

no

change

at

the

gene

expression

level

Fig.

effect

of

rosiglitazone

on

the

secretion

of

complement

as

measured

by

gel

analysis

The

marked

bands

-LRB-

arrows

-RRB-

were

identified

as

One

representative

image

is

shown

A

sample

treated

by

Brefeldin

A

-LRB-

BFA

-RRB-

was

used

to

verify

secretionFig

effect

of

rosiglitazone

on

the

secretion

of

adipsin

as

measured

by

gel

analysis

A

section

of

one

representative

gel

image

is

shown

for

an

individual

adipsin

spot

-LRB-

squares

-RRB-

from

a

low

glucose

and

insulin

control

condition

-LRB-

Basal

-RRB-

b

rosiglitazone

with

low

insulin

and

glucose

condition

-LRB-

Rosi

-RRB-

c

high

glucose

and

insulin

control

condition

-LRB-

IG

-RRB-

and

d

rosiglitazone

with

high

insulin

and

glucose

condition

-LRB-

RosiIG

-RRB-

e

Total

quantity

of

adipsin

in

the

culture

medium

of

adipocytes

expressed

in

arbitrary

units

-LRB-

AU

-RRB-

Fig.

effect

of

rosiglitazone

on

the

secretion

of

adiponectin

as

measured

by

western

blotting

Data

are

expressed

as

mean

SD

-LRB-

n

-RRB-

and

one

representative

western

blot

image

is

shown

p

compared

with

Basal

condition

-LRB-

t

test

-RRB-

p

compared

with

IG

condition

-LRB-

t

test

-RRB-

Basal

low

glucose

and

insulin

control

condition

Rosi

rosiglitazone

with

low

insulin

and

glucose

condition

IG

high

glucose

and

insulin

control

condition

RosiIG

rosiglitazone

with

high

insulin

and

glucose

condition

General

confirmation

of

the

microarray

data

by

quantitative

real-time

RT-PCR

analysis

To

confirm

our

microarray

data

we

checked

the

expression

of

genes

that

encode

secreted

proteins

or

are

related

to

lipid

metabolism

by

Q-PCR

using

as

reference

was

chosen

because

of

its

constant

expression

behaviour

on

the

arrays

A

gene

was

selected

as

target

gene

because

either

itself

or

its

family

member

showed

changed

expression

based

on

microarray

data

The

comparison

showed

a

strong

correlation

between

array

data

and

Q-PCR

data

-LRB-

ESM

Fig.

-RRB-

with

Pearson

correlation

coefficients

of

and

for

Rosi/basal

and

RosiIG/IG

respectively

The

effect

of

rosiglitazone

on

lipolysis

We

assessed

the

glycerol

level

in

the

culture

medium

as

a

measure

of

the

lipolysis

of

adipocytes

RosiIG

treatment

significantly

increased

the

release

of

glycerol

from

mature

adipocytes

whereas

glycerol

release

was

significantly

decreased

by

Rosi

treatment

-LRB-

Fig.

-RRB-

Fig.

effect

of

rosiglitazone

on

the

culture

medium

glycerol

levels

Data

are

expressed

as

mean

SD

-LRB-

n

-RRB-

p

compared

with

IG

condition

-LRB-

t

test

-RRB-

p

compared

with

Basal

condition

-LRB-

t

test

-RRB-

Basal

low

glucose

and

insulin

control

condition

Rosi

rosiglitazone

with

low

insulin

and

glucose

condition

IG

high

glucose

and

insulin

control

condition

RosiIG

rosiglitazone

with

high

insulin

and

glucose

condition

Discussion

As

a

PPARG

agonist

rosiglitazone

stimulates

adipogenesis

which

is

accompanied

by

an

increasing

lipid

content

of

the

differentiating

cells

However

our

results

showed

that

rosiglitazone

does

not

induce

an

increase

of

the

lipid

content

but

decreases

it

in

mature

adipocytes

at

high

insulin/glucose

level

The

pathway

analysis

based

on

transcriptome

data

points

out

that

an

altered

energy

metabolism

may

underlie

this

phenomenon

Although

the

level

of

insulin/glucose

has

some

influence

the

effect

of

rosiglitazone

on

the

transcriptome

was

clearly

dominant

The

most

significantly

regulated

pathways

by

rosiglitazone

in

adipocytes

were

fatty

acid

oxidation

the

TCA

cycle

and

oxidative

phosphorylation

Besides

an

upregulation

of

fatty

acid

catabolism

pathways

our

data

indicate

that

rosiglitazone

encourages

adipocytes

to

utilise

fatty

acids

for

energy

production

and

spares

glucose

and

amino

acids

by

enhancing

glycogen

synthesis

and

inhibiting

amino

acid

catabolism

pathways

This

is

in

line

with

the

upregulation

of

and

the

downregulation

of

expression

The

increase

in

the

level

of

is

correlated

with

a

shift

in

substrate

utilisation

in

favour

of

lipids

as

the

dominant

metabolic

fuel

-LSB-

-RSB-

is

involved

in

fatty

acid

synthesis

but

it

has

also

been

shown

that

deficiency

activates

metabolic

pathways

that

promote

beta-oxidation

and

decrease

lipogenesis

-LSB-

-RSB-

Our

conclusions

regarding

energy

metabolism

are

consistent

with

recent

studies

Mitochondrial

changes

have

been

shown

in

response

to

TZD

treatment

in

adipocytes

-LSB-

-RSB-

human

adipocytes

in

vitro

-LSB-

-RSB-

and

mouse

adipocytes

in

vivo

-LSB-

-RSB-

These

studies

show

that

TZDs

including

rosiglitazone

upregulate

fatty

acid

oxidation

by

increasing

mitochondrial

protein

and

gene

expression

but

not

in

a

trans-differentiation

manner

-LSB-

-RSB-

This

has

been

confirmed

by

direct

measurement

of

labelled

palmitic

acid

oxidation

-LSB-

-RSB-

or

indirect

calorimetric

calculation

-LSB-

-RSB-

We

also

found

that

among

the

genes

involved

in

protein

biosynthesis

more

than

of

those

upregulated

by

rosiglitazone

were

genes

encoding

mitochondrial

ribosomal

proteins

This

is

consistent

with

an

increase

of

mitochondrial

biogenesis

and

remodelling

-LSB-

-RSB-

We

speculate

that

the

maintenance

of

increased

mitochondrial

mass

and

function

utilises

-LRB-

part

of

-RRB-

the

energy

that

the

cell

no

longer

needs

for

lipid

synthesis

under

rosiglitazone

treatment

A

decrease

in

lipogenesis

pathways

accompanies

the

increased

fatty

acid

catabolism

pathways

caused

by

rosiglitazone

in

mature

adipocytes

as

shown

by

decreased

expression

of

genes

encoding

triacylglycerol

synthesis

enzymes

and

the

genes

encoding

lipase

The

downregulation

of

Fasn

by

TZD

has

also

been

observed

in

brown

adipocytes

-LSB-

-RSB-

-LRB-

-RRB-

is

an

important

protein

for

efficient

lipid

droplet

formation

-LSB-

-RSB-

The

downregulation

of

its

gene

expression

supports

a

decrease

of

lipid

accumulation

The

upregulation

of

adipocyte

differentiation

related

protein

gene

-LRB-

Adfp

-RRB-

which

encodes

a

lipid

droplet-associated

protein

seems

to

conflict

with

the

decreased

lipogenesis

observed

by

us

-LRB-

Table

-RRB-

However

in

mature

adipocytes

ADFP

is

spatially

associated

with

lipid

droplets

subjected

to

lipolysis

and

is

suggested

to

play

a

role

in

lipolysis

-LSB-

-RSB-

Furthermore

the

protein

level

of

ADFP

in

mature

adipocytes

is

not

determined

by

the

mRNA

expression

but

by

the

amount

of

micro-lipid

droplets

-LSB-

-RSB-

Therefore

the

increase

in

Adfp

expression

might

also

be

interpreted

as

being

in

agreement

with

upregulated

lipolysis

The

pentose

phosphate

pathway

provides

NADPH

to

be

used

in

reductive

biosynthesis

including

fatty

acid

biosynthesis

-LSB-

-RSB-

Rosiglitazone

enhances

the

pentose

phosphate

pathway

but

inhibits

fatty

acid

biosynthesis

while

stimulating

other

NADPH-dependent

pathways

such

as

glutathione

detoxification

cholesterol

and

steroid

biosynthesis

and

prostaglandin

metabolism

-LRB-

carbonyl

reductase

-RRB-

The

excess

of

NADPH

may

be

a

driving

force

behind

the

upregulation

of

these

pathways

by

rosiglitazone

The

present

study

indicates

that

in

mature

adipocytes

rosiglitazone

enhances

fatty

acid

catabolism

increases

lipolysis

and

reduces

lipogenesis

pathways

This

may

lead

to

a

net

decrease

of

lipid

content

of

the

cells

Indeed

we

observed

increased

lipolysis

expressed

in

terms

of

an

increased

glycerol

level

in

the

medium

and

a

decreased

lipid

content

of

the

cells

under

RosiIG

treatment

This

is

in

line

with

the

increased

lipolysis

effected

by

rosiglitazone

in

a

recent

study

on

rat

white

adipose

tissue

-LSB-

-RSB-

We

did

not

observe

increased

lipolysis

under

Rosi

treatment

where

in

fact

the

glycerol

level

was

decreased

rather

than

increased

The

difference

implies

that

the

insulin/glucose

level

alters

the

effect

of

rosiglitazone

It

is

likely

that

at

low

insulin/glucose

level

the

starved

adipocytes

use

glycerol

as

an

energy

source

rather

than

releasing

it

into

the

medium

However

it

should

be

noted

that

low

insulin/glucose

is

not

a

realistic

situation

in

vivo

when

rosiglitazone

is

administered

The

decreased

lipid

content

is

consistent

with

the

physiological

effects

of

TZDs

in

both

animal

models

-LSB-

-RSB-

and

in

humans

-LSB-

-RSB-

where

TZDs

increase

the

number

of

small

adipocytes

In

adipose

tissue

treated

with

TZDs

mature

adipocytes

can

become

smaller

by

actively

burning

fat

in

parallel

to

the

emerging

of

small

immature

adipocytes

due

to

preadipocyte

differentiation

With

rosiglitazone

mature

adipocytes

take

fatty

acids

for

burning

rather

than

for

storage

This

still

fits

the

fatty

acid

steal

hypothesis

-LSB-

-RSB-

and

can

explain

part

of

the

glucose-lowering

effects

of

rosiglitazone

Rosiglitazone

affects

the

function

of

mature

adipocytes

not

only

with

regard

to

fat

metabolism

but

also

with

regard

to

adipokine

secretion

Despite

the

limitations

of

the

present

study

namely

that

a

mid-size

array

was

used

and

that

the

expression

changes

of

several

genes

could

not

be

detected

due

to

sensitivity

our

data

at

both

the

mRNA

level

and

the

protein

level

confirmed

that

rosiglitazone

represses

adipokine

expression

except

adiponectin

and

Apoe

Since

the

majority

of

adipokines

are

negatively

correlated

with

insulin

sensitivity

-LSB-

-RSB-

the

general

repression

may

correct

the

disturbance

of

insulin

sensitivity

caused

by

obesity

Furthermore

our

transcriptome

analysis

suggests

that

rosiglitazone

induces

a

general

decrease

in

expression

of

genes

encoding

secreted

proteins

This

implies

a

general

mechanism

in

which

energy

redistribution

might

be

involved

We

observed

that

repression

at

the

secretion

level

was

less

significant

than

at

the

gene

expression

level

One

explanation

is

that

the

effect

on

protein

lags

behind

the

effect

on

mRNA

Another

reason

might

be

the

influence

of

insulin

We

have

recently

studied

the

role

of

insulin

in

adipocyte

secretion

-LSB-

-RSB-

We

observed

that

at

a

low

insulin

level

the

secretion

of

proteins

is

already

suppressed

Consequently

the

inhibitory

effect

of

rosiglitazone

on

secretion

is

less

prominent

in

the

low

insulin

condition

In

contrast

to

most

adipokines

adiponectin

is

reversely

correlated

with

obesity

and

its

circulating

level

is

upregulated

by

TZDs

-LSB-

-RSB-

A

functional

PPAR-responsive

element

-LRB-

PPRE

-RRB-

in

the

adiponectin

promoter

could

be

the

reason

for

the

stimulation

-LSB-

-RSB-

We

observed

an

upregulation

only

at

the

secretion

level

but

not

at

gene

expression

level

This

is

in

line

with

the

conclusion

from

another

study

that

although

plasma

levels

of

adiponectin

were

upregulated

by

TZDs

there

was

no

significant

effect

on

adiponectin

gene

expression

-LSB-

-RSB-

Rosiglitazone

is

a

potent

PPARG

agonist

It

seems

logical

to

assume

that

all

these

effects

are

mediated

through

regulation

of

PPARG

Indeed

we

observed

that

rosiglitazone

induced

a

significant

decrease

of

Pparg

expression

in

mature

adipocytes

This

is

in

contrast

to

an

overwhelming

increase

during

adipogenesis

but

it

is

in

line

with

several

reports

-LSB-

-RSB-

With

respect

to

PPARG

downstream

effects

we

found

here

that

two

PPRE-bearing

genes

and

were

downregulated

and

two

other

genes

adiponectin

and

lipoprotein

lipase

remained

unchanged

while

most

PPRE-bearing

genes

-LSB-

-RSB-

such

as

antigen

fatty

acid

binding

protein

adipocyte

and

acyl-coenzyme

A

oxidase

palmitoyl

were

upregulated

by

rosiglitazone

-LRB-

Tables

and

-RRB-

The

effect

of

TZDs

on

in

mature

adipocytes

is

opposite

to

that

in

preadipocytes

-LSB-

-RSB-

and

the

effect

on

is

in

contrast

to

the

reports

in

other

cell

types

-LSB-

-RSB-

It

has

been

known

that

PPARG

acts

with

different

transcriptional

partners

co-repressors

and/or

co-activators

as

a

complex

to

activate

or

repress

the

expression

of

target

genes

-LSB-

-RSB-

Possibly

the

PPARG

complex

is

modulated

by

rosiglitazone

in

mature

adipocytes

in

a

manner

that

is

different

from

that

occurring

in

preadipocytes

and

other

cell

types

In

conclusion

we

have

shown

that

rosiglitazone

in

contrast

to

the

stimulation

of

adipogenesis

in

preadipocytes

reduces

adiposity

of

mature

adipocytes

by

increasing

fatty

acid

catabolism

pathways

In

addition

rosiglitazone

represses

the

expression

and

secretion

of

adipokines

of

mature

adipocytes

Our

findings

suggest

that

the

change

of

adiposity

as

seen

in

vivo

-LSB-

-RSB-

may

reflect

a

shift

in

balance

between

the

effects

of

TZDs

on

preadipocytes

and

those

on

mature

adipocytes

while

the

changes

in

circulating

adipokine

levels

primarily

result

from

an

effect

on

mature

adipocytes

These

aspects

all

contribute

to

the

therapeutic

effects

of

TZDs

Electronic

Supplementary

Material

Below

is

the

link

to

the

electronic

supplementary

material

Table

Q-PCR

primer

sequences

-LRB-

DOC

kb

-RRB-

Fig.

Comparison

between

microarray

and

quantitative

real

time

RT-PCR

analysis

The

mean

of

the

fold-change

is

shown

n

for

array

n

for

RT-PCR

The

lines

at

and

fold

changes

indicate

thresholds

of

differentially

expression

-LRB-

DOC

kb

-RRB-

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

adipokine
adipose tissue
energy metabolism
lipogenesis
lipolysis
mature adipocytes
microarray
secretion


In Vivo Mechanical Loading Modulates Insulin-Like Growth Factor Binding Protein-2 Gene Expression in Rat Osteocytes Mechanical stimulation is essential for maintaining skeletal integrity . Mechanosensitive osteocytes are important during the osteogenic response . The growth hormone-insulin-like growth factor -LRB- GH-IGF -RRB- axis plays a key role during regulation of bone formation and remodeling . Insulin-like growth factor binding proteins -LRB- IGFBPs -RRB- are able to modulate IGF activity . The aim of this study was to characterize the role of IGFBP-2 in the translation of mechanical stimuli into bone formation locally in rat tibiae . Female Wistar rats were assigned to three groups -LRB- n = 5 -RRB- : load , sham , and control . The four-point bending model was used to induce a single period of mechanical loading on the tibial shaft . The effect on IGFBP-2 mRNA expression 6 hours after stimulation was determined with nonradioactive in situ hybridization on decalcified tibial sections . Endogenous IGFBP-2 mRNA was expressed in trabecular and cortical osteoblasts , some trabecular and subendocortical osteocytes , intracortical endothelial cells of blood vessels , and periosteum . Megakaryocytes , macrophages , and myeloid cells also expressed IGFBP-2 mRNA . Loading and sham loading did not affect IGFBP-2 mRNA expression in osteoblasts , bone marrow cells , and chondrocytes . An increase of IGFBP-2 mRNA-positive osteocytes was shown in loaded -LRB- 1.68-fold -RRB- and sham-loaded -LRB- 1.35-fold -RRB- endocortical tibial shaft . In conclusion , 6 hours after a single loading session , the number of IGFBP-2 mRNA-expressing osteocytes at the endosteal side of the shaft and inner lamellae was increased in squeezed and bended tibiae . Mechanical stimulation modulates IGFBP-2 mRNA expression in endocortical osteocytes . We suggest that IGFBP-2 plays a role in the lamellar bone formation process . Mechanical stimulation is important for the maintenance of the skeleton of humans and other animals -LSB- 1 -- 9 -RSB- . Bone serves as a structural support for the body and has the ability to modify its architecture throughout life . Mechanical stimulation regulates bone mass and architecture according to Frost 's mechanostat hypothesis . Physical activity results in increased bone formation and decreased bone resorption , whereas inactivity has the opposite effect -LSB- 10 , 11 -RSB- . The growth hormone-insulin-like growth factor -LRB- GH-IGF -RRB- axis plays an important role in the regulation of bone remodeling . Of the insulin-like growth factors -LRB- IGFs -RRB- , IGF-I is predominantly expressed in rat bone and IGF-II is predominantly expressed in human bone -LSB- 12 -- 14 -RSB- . IGFs are involved in bone formation after mechanical stimulation . Mechanical loading upregulates IGF-I mRNA expression in osteocytes , as shown in rat vertebra by Lean and colleagues using the caudal vertebra compression model -LSB- 15 -RSB- . Northern blot analysis also showed enhanced periosteal IGF-I mRNA expression after mechanical stimulation -LSB- 16 -RSB- . Besides an increase in IGF-I mRNA synthesis , Bravenboer and colleagues reported upregulation of IGF-I protein concentration in bone after mechanical stimulation using additional weight bearing in running rats -LRB- rat-with-backpack -RRB- -LSB- 17 -RSB- . As in other tissues , the autocrine and paracrine actions of IGFs in bone are modulated by a family of six structurally related binding proteins , the IGF binding proteins -LRB- IGFBPs -RRB- . IGFBPs can be synthesized by osteoblasts -LSB- 18 , 19 -RSB- and are capable of modulating the effects of IGFs by potentiating or inhibiting their action ; some IGFBPs also have IGF-independent actions that can either inhibit or stimulate cellular function . Since rat osteoblasts do not express IGFBP-1 -LSB- 18 , 20 , 21 -RSB- , it is most likely that IGFBP-1 does not play a significant role during bone formation after mechanical stimulation in rats . Although IGFBP-2 is the major IGFBP synthesized by rat osteoblastic cells -LSB- 18 -RSB- , a role for IGFBP-2 during mechanical loading has not been established yet . IGFBP-3 has both inhibitory and stimulatory effects on bone cells . IGFBP-4 , which is locally produced by osteoblasts , has an inhibitory effect on bone metabolism , whereas locally synthesized IGFBP-5 by osteoblasts has a stimulatory effect -LSB- 18 , 22 , 23 -RSB- . However , the effects of IGFBP-4 on osteoblasts are IGF-I-dependent , whereas IGFBP-5 probably acts as a store for IGFs in bone tissue -LSB- 18 , 22 , 23 -RSB- . IGFBP-6 is expressed in rat osteoblasts -LSB- 24 -RSB- . In conclusion , IGFBP-2 is the most abundant IGFBP in rat osteoblasts ; however , the role of IGFBP-2 during bone formation after mechanical stimulation in vivo has not been determined yet . We hypothesized that IGFBP-2 mRNA expression level might be changed in bone cells during bone formation after mechanical stimulation . The aim of this study , therefore , was to determine the localization of IGFBP-2 mRNA in the cortical tibial shaft after mechanical stimulation . To this end , we induced a single period of mechanical loading using the four-point bending model of Forwood and Turner -LSB- 1 , 2 , 7 -RSB- . This resulted in bone formation in the rat tibia 5 -- 8 days after stimulation -LSB- 25 -RSB- . We developed an in situ hybridization method especially for bone tissue to detect the local osteogenic response at the cellular level 6 hours after a single period of dynamic loading . Materials and Methods Animals Fifteen female 12-week-old Wistar rats -LRB- 235 ± 12 g ; Harlan , Zeist , The Netherlands -RRB- were randomly assigned to three weight-matched groups -LRB- n = 5/group -RRB- : load , sham , and control . The animal experiment was in accordance with the governmental guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of the VU University Medical Center -LRB- Amsterdam , The Netherlands -RRB- . In vivo Mechanical Loading The right tibiae underwent `` mediolateral '' loading -LRB- load -RRB- , sham loading -LRB- sham , in which the opposed pads were placed at the inner position , 11 mm apart -RRB- , or no loading -LRB- control -RRB- using the four-point bending system of Forwood and Turner -LSB- 1 , 7 -RSB- . Since loading will result in bending and squeezing of the tibia and sham loading only in squeezing of the tibia , the sham group was used as a control for the load group . The left tibiae served as contralateral controls . The four-point bending model -LSB- 25 -RSB- was used to generate a single period of dynamic loading of the right tibia in rats in vivo in order to detect acute changes of IGFBP-2 mRNA locally in bone tissue after stimulation by mechanical stress . The rats were subjected to a single episode of loading comprising 300 cycles -LRB- 2 Hz -RRB- using a peak magnitude of 60 N. Forwood and Turner showed that a single loading session resulted in bone formation in the rat tibia 5 -- 8 days after stimulation -LSB- 25 -RSB- . Characterization of the in vivo strain using the four-point bending system was reported by Akhter and colleagues -LSB- 26 -RSB- . Using the four-point bending system at our laboratory , we demonstrated bone formation at the endosteal surface of the rat tibia 15 days after a single loading session with a frequency of 2 Hz during 300 cycles and an applied peak magnitude of 60 N -LSB- 27 -RSB- . The loading experiment was performed under general anesthesia -LRB- 2 % isoflurane in 1 L/min O2 and 2 L/min N2O -RRB- . The rats were killed exactly 6 hours after loading . This time point was based on the literature -LSB- 15 -RSB- , which was confirmed by a time-course pilot experiment at our laboratory using real-time reverse-transcription polymerase chain reaction analysis -LRB- H. W. van Essen , personal communication -RRB- showing the highest IGF-I mRNA expression 6 hours after loading . Since the actions of IGFs are influenced by their IGFBPs , we examined IGFBP-2 mRNA expression at 6 hours after loading . The tibiae were dissected and immediately fixed in 4 % -LRB- w/v -RRB- paraformaldehyde -LRB- buffered in phosphate-buffered saline -LSB- PBS -RSB- , pH 7.4 -RRB- at 4 °C for 24 hours . Tissue After fixation , the tibiae were decalcified in 10 % ethylenediaminetetraacetic acid -LRB- EDTA -RRB- with 0.5 % paraformaldehyde in PBS at 4 °C for 4.5 weeks . Finally , the tibiae were washed in PBS and dehydrated through a series of ethanol and xylene at room temperature and embedded in paraffin . As a positive control , brain tissue was used -LSB- 28 -RSB- . Brains were dissected rostrally to the cerebellum -LRB- interaural coordinate 0 mm -RRB- and the hippocampus -LRB- interaural coordinate 4 mm -RRB- in three coronal blocks and immediately fixed in 4 % -LRB- w/v -RRB- paraformaldehyde -LRB- buffered in PBS , pH 7.4 -RRB- at 4 °C for 24 hours , followed by washing in PBS , dehydration through a series of ethanol and xylene at room temperature , and embedding in paraffin . Reagents All restriction enzymes and modifying enzymes were purchased from Roche Molecular Biochemicals -LRB- Mannheim , Germany -RRB- , as well as digoxigenin-uridine triphosphate -LRB- UTP -RRB- , antidigoxigenin Fab fragments , nitroblue tetrazolium chloride -LRB- NBT -RRB- , 5-bromo-4-chloro-3-indolyl phosphate -LRB- BCIP -RRB- , and blocking reagent . Nylon membranes were purchased from Qiagen -LRB- Hilden , Germany -RRB- . Polyvinyl alcohol was obtained from Aldrich -LRB- Milwaukee , WI -RRB- . Euparal mounting medium was purchased from Chroma Gesellschaft -LRB- Waldeck , Division Chroma , Münster , Germany -RRB- . Silane-coated glass slides were obtained from Sigma-Aldrich -LRB- St. Louis , MO -RRB- . Mouse IGFBP-2 cDNA was kindly provided by Dr. S. L. S. Drop and Dr. J. W. van Neck -LRB- Department of Pediatrics , Sophia Children 's Hospital , Erasmus University , Rotterdam , The Netherlands -RRB- via Dr. S. C. van Buul-Offers -LRB- Department of Metabolic and Endocrine Diseases , University Medical Center , Utrecht , The Netherlands -RRB- . Synthesis of Digoxigenin-Labeled Complementary RNA -LRB- cRNA -RRB- Probes Standard in vitro transcription reactions were carried out using T7 - and Sp6-RNA polymerase with digoxigenin-UTP as a substrate -LSB- 29 -RSB- . cDNA encoding mouse IGFBP-2 cDNA , corresponding to amino acid position 98 -- 258 and showing 95 % homology with the corresponding rat IGFBP-2 cDNA fragment , was used as a template for the synthesis of antisense and sense digoxigenin-labeled RNA probe . The probe was specific for the mRNAs analyzed . The IGBP-2 probe did not show cross-reactivity with different types of mouse tissues -LRB- spleen , thymus , and brain -RRB- -LSB- 28 , 30 , 31 -RSB- . Nonradioactive In Situ Hybridization Serial , longitudinal tibial sections -LRB- 5 μm -RRB- , which were cut in the posterior-anterior direction , and cross-sectional control brain sections -LRB- 5 μm -RRB- were mounted onto RNase-free silane-coated glass slides and dried at 56 °C for at least 3 days . Corresponding sections of the right and left tibia of one rat were mounted on the same glass slide . In situ hybridization was performed on every fiftieth section with a total of five slides per tibia -LRB- i.e. , sections I , II , II , IV , and V -RRB- . All sections were dewaxed , rehydrated , and rinsed in water . The sections were pretreated with 0.2 N HCl for 15 minutes at room temperature , permeabilized in proteinase K -LRB- 15 μg/mL -RRB- for 30 minutes at 37 °C , and subjected to an acetylation treatment -LSB- 32 -RSB- . Sections were rinsed in 2 × SSC -LRB- 0.3 M sodium chloride and 0.03 M sodium citrate -RRB- and kept in this solution until the start of hybridization . Hybridization was performed in a solution containing 50 % formamide , 2 × SSC , 1 × Denhardt 's solution , 250 μg/mL transfer RNA , 480 μg/mL herring sperm RNA , 10 % dextran-sulfate , and the mouse IGFBP-2 digoxigenin-labeled cRNA probe at concentrations of 500 pg/μL -LRB- brain -RRB- and 1,000 pg/μL -LRB- tibia -RRB- . Sections were hybridized overnight at 53 °C . After hybridization , sections were washed with 50 % formamide in 2 × SSC at the hybridization temperature for 30 minutes and treated with RNase A -LRB- 1 unit/mL -RRB- for 30 minutes at 37 °C . Subsequently , sections were rinsed in 2 × SSC , treated with 1 % blocking reagent for 30 minutes , and incubated with sheep antidigoxigenin Fab fragments conjugated with alkaline phosphatase -LRB- 1:1,500 -RRB- overnight at 4 °C . Chromogenesis was performed in the dark with 0.38 mg/mL NBT and 0.19 mg/mL BCIP in the presence of 6 % -LRB- w/v -RRB- polyvinyl alcohol -LSB- 33 -RSB- , resulting in a blue precipitate . Finally , sections were counterstained with nuclear fast red , dehydrated through a series of ethanol , and mounted with Euparal . Sense probes were used to investigate the level of nonspecific binding . Quantification and Statistics First , a semiquantitative screening of all animals per group was performed on the antisense and sense slides to collect the in situ hybridization data . Sections I-V of all tibiae were scored in a semiquantitative manner , which was defined , on a Nikon Eclipse E800 microscope -LRB- Uvikon , Bunnik , The Netherlands -RRB- , as follows : no expression -LRB- 0 -RRB- , low expression -LRB- 1 -RRB- , average expression -LRB- 2 -RRB- , and high expression -LRB- 3 -RRB- . Effects of mechanical loading were expected in the shaft of the tibiae . Therefore , a second quantitative evaluation of the endocortical osteocytes expressing IGFBP-2 mRNA was performed with Lucia G Version 4.82 -LRB- Uvikon -RRB- using coded slides of sections II and III . The defined area of interest , which comprised the loading zone , started below the primary spongiosa at the endosteal side of the shaft following the total length of the shaft to the distal side -LRB- total length maximal 3,852 μm -RRB- . At the endosteal side of the shaft , all osteocytes , which were positioned within 100 μm of the endosteal surface , were included in the area of interest . Within the area of interest , total osteocyte number and total IGFBP-2 mRNA-positive osteocyte number were measured . The paired-samples Student 's t-test and independent samples Student 's t-test were used for statistical analysis using SPSS -LRB- Chicago , IL -RRB- version 9.0 for Windows . P < 0.05 was considered to reflect statistical significance . Photography Brightfield photographs with different magnifications -LRB- objectives × 10 , × 20 , and × 40 -RRB- were made using a Leica microscope -LRB- DM4000B -RRB- with a digital camera -LRB- Leica DC500 -RRB- and Leica software IM50 -LRB- Leica Microsystems , Rijswijk , The Netherlands -RRB- . Results Endogenous IGFBP-2 mRNA Expression in Tissue The control brain showed IGFBP-2 mRNA expression in the choroid plexus -LRB- Fig. 1A -RRB- and the neurons of the medulla oblongata -LRB- data not shown -RRB- . In the internal control , i.e. , the growth plate , IGFBP-2 mRNA was located in chondrocytes of the proliferative and hypertrophic zone -LRB- Fig. 1C -RRB- . In control tibiae , IGFBP-2 mRNA was expressed in osteoblasts , which were situated against the surface of trabecular bone -LRB- Fig. 1C , E -RRB- and the endosteal side of the shaft -LRB- Fig. 1G -RRB- . IGFBP-2 mRNA expression was also observed in some trabecular osteocytes and in cortical osteocytes , which were situated within the first lamella at the endosteal side of the shaft -LRB- Fig. 1G -RRB- . The endocortical osteocytes , which were located within the deeper lamellae -LRB- Fig. 1G -RRB- , and the periosteal osteocytes -LRB- Fig. 1D -RRB- did not express IGFBP-2 mRNA . IGFBP-2 mRNA was also expressed in the intracortical endothelial cells of blood vessels -LRB- Fig. 1G -RRB- and in the periosteum of control tibiae -LRB- Fig. 1D -RRB- . Some cells of the bone marrow , i.e. , megakaryocytes , macrophages , and myeloid cells , also expressed IGFBP-2 mRNA -LRB- Fig. 1E -RRB- . No differences of IGFBP-2 mRNA expression between the right and left tibiae of the control group were observed using semiquantitative analysis . Fig. 1Expression of IGFBP-2 mRNA in rat brain sections -LRB- A , B -RRB- and in longitudinal control -LRB- C-G -RRB- , sham-loaded -LRB- H -RRB- , and loaded -LRB- J -RRB- tibial sections of 12-week-old female Wistar rats . The RNA signal is shown as a blue precipitate . The sections were counterstained with nuclear fast red and visualized under brightfield illumination . The scale bars represent 100 μm -LRB- A , B -RRB- , 50 μm -LRB- D , E , G-J -RRB- , and 100 μm -LRB- C , F -RRB- . -LRB- A -RRB- IGFBP-2 mRNA expression within the choroid plexus of a control rat brain ; red blood cells are negative and do not express IGFBP-2 mRNA . -LRB- B -RRB- Hybridization with representative IGFBP-2 sense probe showed no signal in the choroid plexus . -LRB- C -RRB- IGFBP-2 mRNA expression in the chondrocytes of the growth plate and osteoblasts of the trabecular bone . -LRB- D -RRB- IGFBP-2 mRNA expression in the periosteum of a control tibia ; the osteocytes in the periosteal side of the shaft do not express IGFBP-2 mRNA . -LRB- E -RRB- IGFBP-2 mRNA expression in osteoblasts situated against the surface of normal trabecular bone and IGFBP-2-positive bone marrow cells . -LRB- F -RRB- Hybridization with a corresponding IGFBP-2 sense probe showed no signal in the osteoblasts , chondrocytes , and bone marrow cells of a control tibia . -LRB- G -RRB- IGFBP-2 mRNA expression within the superficial endocortical osteocytes -LRB- indicated by arrowheads -RRB- , osteoblasts , and intracortical endothelial cells of a blood vessel of a control tibia . -LRB- H -RRB- IGFBP-2 mRNA expression in the endocortical osteocytes within multiple layers -LRB- indicated by arrowheads -RRB- and osteoblasts of a sham-loaded tibia . -LRB- J -RRB- IGFBP-2 mRNA expression in the endocortical osteocytes within multiple layers -LRB- indicated by arrowheads -RRB- and osteoblasts of a loaded tibia . Control hybridizations with the corresponding sense RNA probe exhibited no signals in the brain -LRB- Fig. 1B -RRB- and tibia -LRB- Fig. 1F -RRB- . Effect of Mechanical Loading on IGFBP-2 mRNA Expression in Osteocytes of the Tibial Shaft Induced IGFBP-2 mRNA expression was observed in osteocytes within the first lamella and within multiple layers at the endosteal side of the shaft of loaded tibia -LRB- 1.68-fold increase , n = 5 -RRB- -LRB- Fig. 1J -RRB- and sham-loaded tibia -LRB- 1.35-fold increase , n = 5 -RRB- -LRB- Fig. 1H -RRB- in contrast to the control tibia -LRB- n = 10 -RRB- , where IGFBP-2 mRNA expression was only seen within the first lamella of the endosteal side of the shaft -LRB- Fig. 1G -RRB- . The proportion of IGFBP-2 mRNA-positive osteocytes was 25.5 ± 12.6 % -LRB- mean ± standard deviation -LSB- SD -RSB- , n = 5 -RRB- for loaded tibiae and 17.2 ± 10.7 % -LRB- mean ± SD , n = 5 -RRB- for the contralateral control tibiae -LRB- Figs. 1G , J and 2A -RRB- . Sham-loaded tibiae and the contralateral control tibiae showed a proportion of IGFBP-2 mRNA-positive osteocytes of 20.5 ± 7.5 % -LRB- mean ± SD , n = 5 -RRB- and 13.2 ± 4.2 % -LRB- mean ± SD , n = 5 -RRB- , respectively -LRB- Figs. 1H and 2B -RRB- . Mechanical loading significantly increased the number of IGFBP-2 mRNA-synthesizing osteocytes in loaded tibiae -LRB- P = 0.001 -RRB- and in sham-loaded tibiae -LRB- P = 0.031 -RRB- -LRB- Fig. 2A , B -RRB- . No statistical differences of IGFBP-2 mRNA-positive osteocytes were observed between the contralateral controls of the loaded and sham-loaded tibiae -LRB- P = 0.475 -RRB- or between the loaded and sham-loaded tibiae -LRB- P = 0.468 -RRB- . Fig. 2Influence of mechanical loading on IGFBP-2 mRNA expression in osteocytes at the endosteal side of the shaft within individual rat tibiae . Values are expressed as -LRB- A -RRB- percentage IGFBP-2 mRNA-positive osteocytes in control versus contralateral loaded tibiae -LRB- ** P = 0.001 , n = 5 -RRB- and -LRB- B -RRB- percentage IGFBP-2 mRNA positive osteocytes in control versus contralateral sham-loaded tibiae -LRB- * P = 0.031 , n = 5 -RRB- . No statistical differences of IGFBP-2 mRNA-positive osteocytes were observed between the control load and control sham groups -LRB- P = 0.475 -RRB- or between the load and sham groups -LRB- P = 0.468 -RRB- . The lines connect the left and right tibiae of the same rat . The mean values of each group are represented by a bar . Effects of Mechanical Loading on IGFBP-2 mRNA Expression in Osteoblasts , Chondrocytes , and Bone Marrow Cells No differences in IGFBP-2 mRNA expression between loaded , sham-loaded , and control tibiae were observed in the osteoblasts , chondrocytes , and bone marrow cells by semiquantitative screening . No differences in morphology of the cells were observed between groups . Discussion This in vivo study showed an increase in the number of endocortical osteocytes expressing IGFBP-2 mRNA in loaded -LRB- 1.68-fold -RRB- and sham-loaded -LRB- 1.35-fold -RRB- tibiae 6 hours after a single period of mechanical loading with the four-point bending system . The upregulation of IGFBP-2 mRNA expression was restricted to osteocytes at the endosteal side of the shaft and multiple inner lamellae . No difference between IGFBP-2 mRNA expression in the left and right control tibia was observed . We conclude that bending and squeezing of rat tibiae result in an increase of locally synthesized IGFBP-2 mRNA by endocortical osteocytes . We suggest that IGFBP-2 plays an as yet unidentified role in the lamellar bone formation process . We suggest that the IGFBP-2-producing osteocytes of the loaded tibiae are involved in the increased lamellar bone formation after mechanical loading . Forwood and colleagues reported that a single short period of loading using the four-point bending system resulted in an increased lamellar bone formation rate at the endosteal surface of rat tibia -LSB- 25 -RSB- . This has been confirmed in a validation study at our laboratory -LSB- 27 -RSB- . The lamellar bone formation is located at the endosteal side of the shaft , which is identical to the location of newly synthesized IGFBP-2 mRNA after mechanical loading , as demonstrated in this study . IGFBP-2 mRNA expression was restricted to this area and was not observed in the osteocytes at the periosteal side of the shaft . Mechanical loading with an external bending load of 60 N in vivo will result in an increased woven bone formation rate at the periosteal surface as a result of irritation of the periosteum and increased lamellar bone formation rate at the endosteal surface -LSB- 1 -RSB- . We suggest that IGFBP-2 mRNA may be less involved in woven bone formation because the osteocytes at the periosteal side of the shaft did not express IGFBP-2 . This suggests that IGFBP-2 mRNA in the mechanosensitive osteocytes is specifically important for lamellar bone formation . In addition , this study showed that the sham-loaded tibiae have increased IGFBP-2 mRNA production in endocortical osteocytes 6 hours after sham loading . Apparently , besides bending , squeezing alone is also responsible for the upregulation of IGFBP-2 mRNA in the endocortical osteocytes . However , squeezing did not induce lamellar bone formation . Remarkably , there is a large range in the data for the percentage of IGFBP-2 mRNA-positive osteocytes in the contralateral control tibiae of the load group in comparison to the data of the contralateral tibiae of the sham group . This could be caused by the biological variation between the individuals because the animals were randomly divided between the groups and there is no significant statistical difference between those tibiae . For this study we used the four-point bending model of Forwood because this apparatus produces a controlled mechanical strain in the tibia of living rats . An advantage of this approach is that it does not require surgical intervention and allows normal physical activity after the loading session -LSB- 1 , 7 -RSB- . The osteogenic response will occur at a distinct location in bone . Therefore , we used the in situ hybridization technique in order to detect the local osteogenic response at the cellular level . Nonradioactive in situ hybridization is a powerful and sensitive technique to localize gene expression within decalcified rat tibiae . It was demonstrated that the IGFBP-2 probe is specific because IGFBP-2 mRNA expression is present in the choroid plexus and the neurons of the medulla oblongata , as described earlier -LSB- 28 -RSB- , and in the chondrocytes of the proliferative and hypertrophic zone of the growth plate -LSB- 34 -RSB- . Nevertheless , this study has some limitations . The applied load of 60 N is supraphysiological , and 6 hours after a single loading session is too early to demonstrate bone formation . In summary , this study shows an increase of the number of osteocytes at the endosteal side of the shaft and inner lamellae expressing IGFBP-2 mRNA 6 hours after mechanical loading and sham loading in vivo . We conclude that these IGFBP-2-producing osteocytes in the endosteal shaft are mechanosensitive . We suggest that IGFBP-2 plays a role in the lamellar bone formation process .

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

mechanical loading
nonradioactive in situ hybridization
in vivo

four-point bending system
igfbp-2 mrna expression