Tadalafil ameliorates metabolic syndrome‐induced alterations in 

visceral adipose tissue: an experimental study in the rabbit

• Mario Maggi• Sexual Medicine and Andrology Unit

• Dept. Experimental and Clinical Biomedical Sciences• University of Florence

• Italy

Ilaria CellaiPaolo ComeglioFrancesca CorcettoChiara CornioSandra FilippiElena Maneschi

Sexual Medicine & Andrology Unit

Dept. «Mario Serio»University of Florence

Linda Vignozzi

Anatomy and Histology UnitsDepartment of Experimental and Clinical Medicine

Prof.Daniele BaniProf. Barbara VannelliDr. Annamaria Morelli, Dr. Erica Sarchielli

Gastroenterology UnitDepartment «Mario Serio»Dr. Tommaso MelloProf. Andrea Galli

Metabolic syndrome (MetS)

High triglyceridesLow HDL cholesterolHypertensionHyperglycemiaVisceral obesity

BAT dissipates energy in the

form of heat, by metabolizing

fatty acids

Inhibition of PDE5

RhoA/ROCK

0.5% cholesteroland 4% peanuts oil

•High fat diet: HFD

3 6 9 12 weeks0

•Standard diet: control

Filippi S et al., J Sex Med 2009, 6(12):3274-88 Vignozzi et al., Mol cell Endocrinol 2014 25;384:143-54. Vignozzi L et al., J Sex Med. 2011 Jan;8(1):57-77Vignozzi et al., .J of Endocrinol 2012 Jan;212(1):71-84Morelli et al., Prostate. 2012 Sep 19. Morelli et al., J Steroid Biochem Mol Biol. 2012 ;132:80. Maneschi et al., J Endocrinol. 2012 Dec;215(3):347-362..

HyperglycaemiaReduced glucose tolerance (OGTT)HypercholesterolemiaHypertriglyceridemiaHypertensionIncreased visceral fat massDysfunctional VATNASH

o ↓ testosterone level (↓ FSH and LH level) o ↓ prostate, seminal vesicles weight

MetS

Hypogonadotropichypogonadism

Aim of the Study: to investigate the effect of a PDE5 inhibitor, Tadalafil, on metabolic features

in a rabbit model of MetS

0.5% cholesteroland 4% peanuts oil

•High fat diet: HFD

3 6 9 12 weeks0

•Standard diet: control

Filippi S et al., J Sex Med 2009, 6(12):3274-88

Vignozzi et al., Mol cell Endocrinol 2014 25;384:143-54. Vignozzi et al., .J of Endocrinol 2012 Jan;212(1):71-84Morelli et al., Prostate. 2012 Sep 19. Morelli et al., J Steroid Biochem Mol Biol. 2012 ;132:80. Maneschi et al., J Endocrinol. 2012 Dec;215(3):347-362..Vignozzi L et al., J Sex Med. 2011 Jan;8(1):57-77

HyperglycaemiaReduced glucose tolerance (OGTT)HypercholesterolemiaHypertriglyceridemiaHypertensionIncreased visceral fat massDysfunctional VATNASH

o ↓ testosterone level (↓ FSH and LH level) o ↓ prostate, seminal vesicles weight

MetS

Hypogonadotropichypogonadism

Rabbit model of Metabolic Syndrome

•High fat diet + acute Tadalafil: HFD + 1 week Tad

Tadalafil(2mg/kg/day)

P=0.02P<0.0001

RD HFD HFD+Tad1week

Trig

lyce

rides

(mg/

dL)

Morelli et al., 2013, Prostate. 73(4):428-41. Vignozzi et al., unpublished 2014

MetS

HyperglycaemiaReduced glucose tolerance (OGTT)HypercholesterolemiaHypertriglyceridemiaHypertensionIncreased visceral fat massDysfunctional VATNASH

Tadalafil significantly reduced HFD‐induced increase in triglycerides

RD HFD HFD+Tad1week

VAT

(% o

f bod

y w

eigh

t)P=0.004

P=0.011 MetS

Tadalafil significantly reduced HFD‐induced increase in VAT weigth

Morelli et al., 2013, Prostate. 73(4):428-41. Vignozzi et al., unpublished 2014

HyperglycaemiaReduced glucose tolerance (OGTT)HypercholesterolemiaHypertriglyceridemiaHypertensionIncreased visceral fat massDysfunctional VATNASH

VAT expresses high level of both PDE5 and PDE11

Vignozzi et al., unpublished 2014

d.

h.

*

*°

40

60

80

100

Control HFD HFD+Tadalafil

Adi

pocy

tedi

amet

er(μ

m)

RD HFD HFD+Tadalafil

*

0

50

100

150

200

250

300

Control HFD HFD+tadalafil

Hyp

oxyp

robe

posit

ivity

(%

of c

ontr

ol)

RD HFD HFD+Tadalafil

HFD‐induced adipocyte hypertrophy and hypoxia were reduced by Tadalafil

Vignozzi et al., unpublished 2014

Maneschi et al. J of Endocrinol 2012 215, 1-17Maneschi et al., J of Endocrinol 218(2):215-31

**

** P<0.01 vs. all the other groups

mGLUT4

cGLUT4

STAT1

0,00

20,00

40,00

60,00

80,00

100,00

120,00

Control Diet Diet+Tad A

mGLUT4/cGLUT4 %

RD HFD HFD+Tadalafil

Vignozzi et al., unpublished 2014

Maneschi et al. J of Endocrinol 2012 215, 1-17Maneschi et al., J of Endocrinol 218(2):215-31

HFD‐induced reduction of GLUT4 translocation to plasma membrane was normalized byTadalafil

UCP1 immunolocalization in rabbit VAT

10x

Isolation of rPADs from visceral fat

RD

rPAD RD

HFD

rPAD HFD

HFD+TAD

rPAD HFD+TAD

Maneschi et al., J Endocrinol. 2013 Jul 6;218(2):215-31.Maneschi et al., J Endocrinol. 2012 Dec;215(3):347-62.

Isolation of rPADs from visceral fat

RD

rPAD RD

HFD

rPAD HFD

HFD+TAD

rPAD HFD+TAD

10 days

NTDMEM with 4,5 g/L Glucose, P/S, L‐GlutamineFETAL BOVINE SERUM 5%

RD NT10

10 days HFD NT10

10 days HFD+Tad NT10

Vignozzi et al., unpublished 2014

Pre-adipocytes expresses very high level of PDE5 (similar to smooth muscleof corpora cavernosa)

Vignozzi et al., unpublished 2014

A. RD B. HFD C. HFD+tadalafil

**

°°

**

°°

D. E.

10 100 1000 10000

TFAMNRF1

PPARϒC‐1αPPARϒC‐1β

PKG1

SDHB

NDUFS1

GCa1

NDUFB3NDUFB5

CSSLC25A12

GCb1

****

***

**

*

****p<0,0001, ***p<0,001, **p<0,01, *p<0,05 vs HFD

UCP1TMEM26

BMP4

PPARγ

RhoAROCK1ROCK2

*

****

*

**

*

****CIDEA

CD137 ****

RB1HOXC9

mRNA expression in HFD+Tadalafil (% of HFD)

↑Mitochondrial biogenesis

↑GC/PKG pathway

↑↑↑Brown/Brite adipocyte markers

↓White adipocyte markers*

HFDRD

HFD+TADALAFIL

Mitochondrial network morphology and dynamics by using a mitochondria specific probe(MitoTracker)

a. RD b. HFD

c. HFD+ tadalafil d.

0

20

40

60

80

100

120

RD HFD HFD+Tadalafil

Tota

l Mito

chon

dria

l cris

tae

surf

ace/

ou

term

embr

ane

surf

ace

p<0.0001p<0.0001

Mitochondrial ultrastructure by using TEM

0

255a. RD

b. HFD

c. HFD+tadalafil d.

Superoxide production by using dihydroethidium (DHE)

In vitro treatment with tadalafil 100 nM in rPADs from HFD rabbits

HFD

rPAD HFD

Untreated

+KT5823

+Tadalafil

+Tadalafil+KT5823

10 days

KT5823: a selectiveinhibitor of PKG

TadalafilTadalafil(inhibits cGMP degradation)

-

↑↑↑↑↑

+8Br cGMP

+BAY 41-8543

BAY 41-853: a selective sGCactivator

8Br cGMP: a PDE-resistantcGMP analog

a. RD b. HFD c. HFD+ tadalafil

d.

0 10-9 10-8 10-7

100

150

200

250

300

rPAD RDrPAD HFDrPAD HFD + tadalafil

Insulin (M)

3 H-g

luco

se u

ptak

e (%

)

**

Mea

nnu

mbe

rof l

ipid

drop

lets

in rP

AD

Mea

nvo

lum

e (μm

3 ) o

f lip

iddr

ople

tsin

rPA

De. f.

In vitro treatment with tadalafil 100 nM in rPADs from HFD rabbits

Figure 10

Untreated 100nMtadalafilRD rPAD

HFD rPAD

a. b.

c. d.

Mito

chon

dria

l len

ght(

µm)

P<0.0001P<0.0001

RD rPAD Untreated HFD rPAD

HFD rPAD +100nM tadalafil

0

20

40

60

80

100

120

140

RD HFD Tadalafil KT5823 KT+Tadalafil

Tota

l mith

onco

ndria

lcris

tae

surfa

ce/ o

uter

mem

bran

e su

rface

Untreated RD rPAD Untreated +100nM tadalafil + KT 5823 +100nM tadalafil+KT5823

HFD rPAD

P<0.0001

P<0.05

P<0.0001

P<0.05

a. b. c.

d. e. f.

In vitro treatment with tadalafil 100 nM in rPADs from HFD rabbits

Tadalafil stimulatesBrown-like phenotype

0

50

100

150

200

250

control Tadalafil KT5823 Tadlf+KT 8BrcGMP BR5381

UCP1

*

* *

#

0

50

100

150

200

250

control Tadalafil KT5823 Tadlf+KT 8BrcGMP BR5381

TMEM26

*

*****

#

0

50

100

150

200

250

NT10 Tadalafil KT5823 Tadlf+KT 8BrcGMP BR5381

*

*#

CD137

mRN

A/18S

mRN

A/18S

0

50

100

150

200

control Tadalafil KT5823 Tadlf+KT 8BrcGMP BAY 41‐8543

CIDEA*

#

Untreated tadalafil KT 5823 tadalafil+KT5823 8-Br-cGMP BAY 41-8543

HFD rPAD

Untreated tadalafil KT 5823 tadalafil+KT5823 8-Br-cGMP BAY 41-8543

HFD rPAD

Untreated tadalafil KT 5823 tadalafil+KT5823 8-Br-cGMP BAY 41-8543Untreated tadalafil KT 5823 tadalafil+KT5823 8-Br-cGMP BAY 41-8543

HFD rPAD

Open label, single-arm, uncontrolled, single center, clinical trial, on 10 male patients aged ≥18 years, referring to an outpatient facility for the treatment of sexual dysfunction.

Primary objective: to determine the potential effects of chronic tadalafil administration (2.5 mg/daily) on body composition in male subjects with erectile dysfunction. The primary outcomes was the variation of abdominal fat mass, measured with a whole body dual-energy X-ray absorptiometry (DXA-HOLOGIC QDR-1000), Further outcomes included body mass index (BMI) and other measures of fat distribution (waist circumference) and body composition (total fat mass with DXA)

Antonio AversaDavide FrancomanoAndrea Lenzi

Department of Experimental Medicine, Medical Pathophysiology, Food Science and Endocrinology Section, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy

All patients were treated with tadalafil 2.5mg daily, in the early morning, for 8 weeks; the treatment was then stopped, and further follow-up evaluation was performed at week 16

Tadalafil 2.5mg daily

0 8 weeks 16 weeks

Stoptreatment

Washout

Antonio AversaDavide FrancomanoAndrea Lenzi

Department of Experimental Medicine, Medical Pathophysiology, Food Science and Endocrinology Section, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy

Open label, single-arm, uncontrolled, single center, clinical trial, on 10 male patients aged ≥18 years, referring to our outpatient facility for the treatment of sexual dysfunction.

Antonio AversaDavide FrancomanoAndrea Lenzi

Department of Experimental Medicine, Medical Pathophysiology, Food Science and Endocrinology Section, Sapienza University of Rome, Viale Regina Elena 324, 00161 Rome, Italy

*

*

* *

a. b.

c.

Fat (kg) Lean (kg) Total mass (kg)

10

15

5

-5

-15

-10

0

Baseline vs 8 weeks8 weeks vs 16 weeks

*p<0.01

conclusions:• in an animal model of MetS (diet-induced) and in human

subjects, in vivo tadalafil administration is able to decrease visceral adipose tissue (VAT), after short-term exposure (1and 8 weeks, respectively).

• in vivo and in vitro studies demonstrated that tadalafil, via PKG activation in VAT, up-regulates browning-specific genes, including UCP1, and counteracts HFD-associated mitochondrial alterations, suggesting VAT browning