Tadalafil ameliorates metabolic syndrome induced alterations in visceral adipose tissue: an experimental study in the rabbit Mario Maggi Sexual

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

Sexual Medicine & Andrology Unit Dept. «Mario Serio» University of Florence Linda Vignozzi Anatomy and Histology Units Department of Experimental and Clinical Medicine Prof.Daniele Bani Prof. Barbara Vannelli Dr. Annamaria Morelli, Dr. Erica Sarchielli Ilaria Cellai Paolo Comeglio Francesca Corcetto Chiara Cornio Sandra Filippi Elena Maneschi Gastroenterology Unit Department «Mario Serio» Dr. Tommaso Mello Prof. Andrea Galli

High triglycerides Low HDL cholesterol Hypertension Hyperglycemia Visceral obesity Metabolic syndrome (MetS)

BAT dissipates energy in the form of heat, by metabolizing fatty acids

Inhibition of PDE5 RhoA/ROCK

Aim of the Study: to investigate the effect of a PDE5 inhibitor, Tadalafil, on metabolic features in a rabbit model of MetS MetS Hyperglycaemia Reduced glucose tolerance (OGTT) Hypercholesterolemia Hypertriglyceridemia Hypertension Increased visceral fat mass Dysfunctional VAT NASH 0 Standard diet: control High fat diet: HFD 0.5% cholesterol and 4% peanuts oil 3 6 9 12 weeks Hypogonadotropic hypogonadism o testosterone level ( FSH and LH level) o prostate, seminal vesicles weight 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):57-77 Vignozzi et al.,.j of Endocrinol 2012 Jan;212(1):71-84 Morelli 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..

Rabbit model of Metabolic Syndrome MetS Hyperglycaemia Reduced glucose tolerance (OGTT) Hypercholesterolemia Hypertriglyceridemia Hypertension Increased visceral fat mass Dysfunctional VAT NASH 0 Standard diet: control High fat diet: HFD 3 6 9 12 weeks 0.5% cholesterol and 4% peanuts oil Tadalafil (2mg/kg/day) Hypogonadotropic hypogonadism o testosterone level ( FSH and LH level) o prostate, seminal vesicles weight High fat diet + acute Tadalafil: HFD + 1 week Tad Vignozzi et al., Mol cell Endocrinol 2014 25;384:143-54. Vignozzi et al.,.j of Endocrinol 2012 Jan;212(1):71-84 Morelli 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 Filippi S et al., J Sex Med 2009, 6(12):3274-88

Tadalafil significantly reduced HFD induced increase in triglycerides P<0.0001 P=0.02 MetS Triglycerides (mg/dl) Hyperglycaemia Reduced glucose tolerance (OGTT) Hypercholesterolemia Hypertriglyceridemia Hypertension Increased visceral fat mass Dysfunctional VAT NASH RD HFD HFD+Tad1week Morelli et al., 2013, Prostate. 73(4):428-41. Vignozzi et al., unpublished 2014

Tadalafil significantly reduced HFD induced increase in VAT weigth P=0.004 P=0.011 MetS VAT (% of body weight) Hyperglycaemia Reduced glucose tolerance (OGTT) Hypercholesterolemia Hypertriglyceridemia Hypertension Increased visceral fat mass Dysfunctional VAT NASH RD HFD HFD+Tad1week Morelli et al., 2013, Prostate. 73(4):428-41. Vignozzi et al., unpublished 2014

VAT expresses high level of both PDE5 and PDE11 Vignozzi et al., unpublished 2014

HFD induced adipocyte hypertrophy and hypoxia were reduced by Tadalafil d. Adipocytediameter (μm) 100 80 60 40 Control RD HFD HFD+Tadalafil h. Hypoxyprobepositivity (% of control) 300 250 200 150 100 50 0 Control RD HFD HFD+Tadalafil HFD+tadalafil Maneschi et al. J of Endocrinol 2012 215, 1-17 Maneschi et al., J of Endocrinol 218(2):215-31 Vignozzi et al., unpublished 2014

HFD induced reduction of GLUT4 translocation to plasma membrane was normalized by Tadalafil 120,00 mglut4/cglut4 % 100,00 80,00 60,00 40,00 20,00 0,00 Control RD HFD Diet HFD+Tadalafil Diet+Tad A mglut4 cglut4 STAT1 P<0.01 vs. all the other groups Maneschi et al. J of Endocrinol 2012 215, 1-17 Maneschi et al., J of Endocrinol 218(2):215-31 Vignozzi et al., unpublished 2014

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 NT DMEM with 4,5 g/l Glucose, P/S, L Glutamine FETAL BOVINE SERUM 5% rpad RD 10 days RD NT10 HFD rpad HFD 10 days HFD NT10 HFD+TAD rpad HFD+TAD 10 days HFD+Tad NT10 Vignozzi et al., unpublished 2014

Pre-adipocytes expresses very high level of PDE5 (similar to smooth muscle of corpora cavernosa) Vignozzi et al., unpublished 2014

A. RD B. HFD C. HFD+tadalafil D. E.

TFAM NRF1 CS SLC25A12 NDUFB5 NDUFB3 NDUFS1 SDHB GCb1 GCa1 PKG1 ROCK2 ROCK1 RhoA HOXC9 RB1 PPARγ PPARϒC 1β PPARϒC 1α BMP4 CD137 TMEM26 UCP1 CIDEA Mitochondrial biogenesis GC/PKG pathway White adipocyte markers p<0,0001, p<0,001, p<0,01, p<0,05 vs HFD 10 100 1000 10000 mrna expression in HFD+Tadalafil (% of HFD) Brown/Brite adipocyte markers

Mitochondrial network morphology and dynamics by using a mitochondria specific probe (MitoTracker) RD HFD HFD+TADALAFIL

Mitochondrial ultrastructure by using TEM a. RD b. HFD c. HFD+ tadalafil d. Total Mitochondrial cristae surface/ outer membrane surface 120 100 80 60 40 20 p<0.0001 p<0.0001 0 RD HFD HFD+Tadalafil

Superoxide production by using dihydroethidium (DHE) a. RD 255 0 b. HFD c. HFD+tadalafil d.

In vitro treatment with tadalafil 100 nm in rpads from HFD rabbits Untreated 10 days HFD +Tadalafil rpad HFD +Tadalafil +KT5823 +KT5823 BAY 41-853: a selective sgc activator Tadalafil (inhibits cgmp degradation) +8Br cgmp +BAY 41-8543 KT5823: a selective inhibitor of PKG - 8Br cgmp: a PDE-resistant cgmp analog

In vitro treatment with tadalafil 100 nm in rpads from HFD rabbits a. RD b. HFD c. HFD+ tadalafil d. e. f. 300 250 200 150 100 rpad RD rpad HFD rpad HFD + tadalafil 3 H-glucose uptake (%) 0 10-9 10-8 10-7 Insulin (M) Mean number of lipid droplets in rpad Mean volume (μm 3 ) of lipid droplets in rpad

Figure 10 a. RD rpad c. b. Untreated HFD rpad d. P<0.0001 Mitochondrial lenght (µm) P<0.0001 RD rpad HFD rpad +100nM tadalafil Untreated 100nMtadalafil HFD rpad

a. b. c. d. e. f. 140 Total mithoncondrialcristae surface / outer membrane surface 120 100 80 60 40 20 P<0.0001 P<0.0001 P<0.05 P<0.05 0 Untreated RD RD rpad Untreated HFD +100nM Tadalafil tadalafil + KT KT5823 5823 +100nM KT+Tadalafil tadalafil+kt5823 HFD rpad

In vitro treatment with tadalafil 100 nm in rpads from HFD rabbits Tadalafil stimulates Brown-like phenotype mrna/18s 250 200 150 100 50 UCP1 # 250 200 150 100 50 TMEM26 # 0 0 control Tadalafil KT5823 Tadlf+KT 8BrcGMP BR5381 Untreated tadalafil KT 5823 tadalafil+kt5823 8-Br-cGMP BAY 41-8543 HFD rpad control Untreated Tadalafil tadalafil KT KT5823 tadalafil+kt5823 Tadlf+KT 8-Br-cGMP 8BrcGMP BAY BR5381 41-8543 HFD rpad 250 200 CD137 200 CIDEA mrna/18s 150 100 # 150 100 # 50 50 0 Untreated tadalafil tadalafil+kt5823 8-Br-cGMP BAY 41-8543 NT10 Tadalafil KT5823 Tadlf+KT 8BrcGMP BR5381 HFD rpad 0 control Tadalafil KT5823 Tadlf+KT 8BrcGMP BAY 41 8543 Untreated tadalafil KT 5823 tadalafil+kt5823 8-Br-cGMP BAY 41-8543

Antonio Aversa Davide Francomano Andrea 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 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 Aversa Davide Francomano Andrea 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 0 8 weeks 16 weeks Tadalafil 2.5mg daily Stop treatment Washout

Antonio Aversa Davide Francomano Andrea 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.

a. b. 15 10 5 0-5 -10-15 Fat (kg) Lean (kg) Total mass (kg) c. p<0.01 Baseline vs 8 weeks 8 weeks vs 16 weeks

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 (1 and 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