Received: 4 January 2018
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Accepted: 30 April 2018
DOI: 10.1002/jcp.26796
RAPID COMMUNICATION
Chronic phosphodiesterase type 5 inhibition has beneficial effects on subcutaneous adipose tissue plasticity in type 2 diabetic mice Daniela Fiore1 Andrea Lenzi1
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Daniele Gianfrilli1
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Andrea M. Isidori1
1 Department of Experimental Medicine, Sapienza University, Rome, Italy 2
Department of Anatomical, Histological, Forensic, and Orthopaedic Sciences, Sapienza University, Rome, Italy
Silvia Cardarelli1
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Fabio Naro2
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Mary A. Venneri1
Different adipose tissue (AT) depots are associated with multiple metabolic risks. Phosphodiesterase type 5 (PDE5) is involved in adipocyte physiology and PDE5 inhibition may affect adipogenesis and ameliorate white AT quality. The aim of this study is to investigate the distribution of AT and the composition of the stroma‐
Correspondence Mary Anna Venneri, Ph.D., Department of Experimental Medicine, Sapienza University of Rome, Viale del Policlinico 155, Rome 00161, Italy. Email:
[email protected] Funding information Ministero dell’Istruzione, dell’Università e della Ricerca, Grant/Award Number: 2015ZTT5KB
vascular fraction (SVF) of subcutaneous AT (SAT) in type 2 diabetic mice after prolonged treatment with a PDE5 inhibitor, Sildenafil. 18 db/db mice were treated with Sildenafil or vehicle for 12 weeks. AT distribution was monitored and SAT was processed for isolation of SVF by flow cytometry. Sildenafil induced an overall reduction in AT, mainly in visceral AT (VAT), compared with SAT. In Sildenafil‐treated mice, the mean change in body weight from baseline positively correlated with VAT, but not with SAT. Characterization of SVF of SAT showed an increase in the frequency of M2 macrophages and endothelial cells in treated mice. Sildenafil improved the maintenance of SAT homeostasis and distribution. KEYWORDS
adipocyte progenitors, endothelial cells, macrophages, phosphodiesterase type 5, sildenafil, subcutaneous adipose tissue, type 2 diabetes
1 | IN TR OD UTI O N
promote AT dysfunction and impair glucose tolerance. A delicate balance of polarized populations of macrophages is necessary to
The distribution of adipose tissue (AT) into subcutaneous (SAT) and
maintain a healthy AT (Sun, Kusminski, & Scherer, 2011). Identifying
visceral (VAT) depots is closely linked to the risk of metabolic
factors that trigger the phenotypic switch from M1 to M2 may be
disease. The accumulation of VAT is strongly associated with
beneficial to preserve adequate adipocyte function. Vascularization is
metabolic dysfunction and cardiovascular disease (Fox et al., 2007).
also critical for the development and homeostasis of AT by providing
In contrast, elevated SAT has a lower or even an inverse correlation
oxygen and nutrients. AT‐associated endothelial cells (ECs) are key
with disease risk and has been associated with improvements in
players for a healthy adipocyte microenvironment (Cao, 2007). AT
plasma lipid profile, insulin sensitivity, blood pressure, and
growth can occur through two distinct mechanisms: an increase in
atherosclerosis (Manolopoulos, Karpe, & Frayn, 2010; McLaughlin,
the size of existing adipocytes (hypertrophy) or an increase in the
Lamendola, Liu, & Abbasi, 2011).
number of adipocytes (hyperplasia). New adipocytes are derived
Increased cytokine production from M1 macrophages and/or
from adipocyte progenitor (AP) cells within the stroma of all white
reduced anti‐inflammatory signals from the M2 macrophages
adipose depots (Berry, Jeffery, & Rodeheffer, 2014). AP abundance, contributing toward a depot’s ability to maintain a pool of small,
Abbreviations: AP, adipocyte progenitor; AT, adipose tissue; EC, endothelial cell; PDE5, phosphodiesterase type 5; PDE5i, PDE5 inhibitor; SAT, subcutaneous adipose tissue; SVF, stroma‐vascular fraction; VAT, visceral adipose tissue.
J Cell Physiol. 2018;1–7.
functional, new adipocytes, promotes hyperplasia rather than hypertrophy (Jeffery et al., 2016).
wileyonlinelibrary.com/journal/jcp
© 2018 Wiley Periodicals, Inc.
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Phosphodiesterase type 5 (PDE5) inhibitors (PDE5is) are selective
tissues were minced with scissors until homogeneous. One milliliter
blockers of the cyclic guanosine monophosphate (cGMP)‐hydrolyzing
CollagenaseD (1.5 U/ml; Roche Biochemicals) with dispase II (2.4 U/ml;
enzyme, whose expression is increased in cardiac hypertrophy. Beyond
Roche Biochemicals, Mannheim, Germany), supplemented in total
the vascular activity, considerign that these drugs are FDA approved
10 mM CaCl2, was applied to each gram of homogenized tissue. The
for erectile dysfunction and pulmonary hypertension, PDE5is show
preparation was incubated at 37°C for 2 hr, washed, and cells were
cardio‐protective effects in patients with type 2 diabetes (T2DM;
collected by centrifugation at 200 relative centrifugal force for 10 min.
Giannetta et al., 2014, 2012). The PDE5i sildenafil also improves
The floating adipocyte layer and the supernatant were removed, and the
insulin sensitivity (Ramirez et al., 2015) and metabolic control
remaining stromovascular cells were passed through a 40 μm cell
(Mandosi et al., 2015). Recently, it was proven that it mitigates
strainer before staining for flow cytometry.
M1‐type macrophage tissue infiltration in diabetic mice (Venneri et al., 2015). The metabolic effects of PDE5is are extended to AT function. In
2.3 | Flow cytometry
vitro and in vivo animal models show that PDE5is may affect adipogenesis and ameliorate white AT quality (Ayala et al., 2007; Handa et al., 2011; Mitschke et al., 2013; Zhang et al., 2010). We recently demonstrated that Sildenafil treatment in human and murine models of diabetes improves VAT, targeting SIRT1 (Sirtuin 1) through a modulation of miR‐22‐3p expression. Intriguingly, M2 macrophages and EC frequency as well as adipocyte AP were increased, ameliorating AT quality (Fiore, Gianfrilli, et al., 2016). To the best of our knowledge, the specific effects of PDE5i on SAT have not been explored. The aim of the current study is to investigate SAT modification induced by PDE5i in diabetic mice.
SVF cells were incubated with primary antibodies using standard methods. Cells were processed for flow cytometry as previously described (Fiore, Judson, et al., 2016). We used the following monoclonal primary antibodies: anti‐CD31 (BD Pharmingen, San Diego, CA) anti‐CD45 (BD), anti‐Sca1 (eBioscience, Thermo Fisher Scientific, Waltham, MA), anti‐F4/80 (eBioscience), and anti‐Mrc1 (R&D System, Minneapolis, MN). Cells were stained with propidium iodide (1 μg/ml) and Hoechst 33,342 (2.5 μg/ml), and resuspended at ~1 × 107 cells/ml density. All samples were analyzed using a CyAn™ ADP cytometer (Dako, Glostrup, Denmark). The biexponential analysis was performed using and FlowJo X (Treestar) software.
2 | MATERIALS AND METHODS
2.4 | Quantitative RT‐PCR
2.1 | Animals
RNA was isolated from VAT and SAT using the Aurum total RNA mini kit (Bio‐Rad) according to the manufacturer’s instructions. Extraction was
Eighteen male BKS.Cg‐Dock7m+/+LeprdbJ (db/db) mice at 10 weeks of
followed by a DNAase digestion step to remove any contaminating
age were randomized to 12 weeks of sildenafil or vehicle treatment
genomic DNA. cDNA was synthesized using the high‐capacity iScript
(9 db/db + vehicle and 9 db/db + sildenafil). Sildenafil citrate (10 mg/kg,
Reverse Transcription kit (Bio‐Rad) following the manufacturer’s instruc-
sildenafil, viagra; Pfizer, New York, NY) was administered by an oral
tions (High‐Capacity cDNA Reverse Transcription kit; Bio‐Rad). The total
gavage. Mice were obtained from the Charles River Laboratory and
cDNA pool obtained served as the template for subsequent polymerase
were maintained in a pathogen‐free facility. All experiments were
chain reaction (PCR) amplification in real‐time (RT)‐PCR using CFX
performed in accordance with Italian law (D.L. 2010/63EU) and the
Connect (Bio‐RAD, Hercules, CA) and data analysis was performed using
study was approved by the Sapienza University’s Animal Research
CFX Manager™ Software provided by Bio‐Rad. Data are expressed as
Ethics Committee and by the Italian Ministry of Health (165/2016‐PR).
fold‐change in expression levels. The relative expression levels were
The appropriate vehicle was administered to db/db + vehicle mice.
normalized to GAPDH mRNA. The following primer pairs were used for
Fasting blood glucose, body weight, and food consumption were
amplification: PDE5, forward: GGAGGAGAATACTGGCAAGA, reverse:
monitored in all animals. Baseline and after sildenafil treatment, blood
GATGCATGGTAAGACAGGAC; CCL2, forward: GTTGGCTCAGCCAG
glucose and triglycerides were assessed using a Siemens Advia 1800
ATGCA, reverse: AGCCTACTCATTGGGATCATCTTG; IL‐10, forward:
Analyzer (Siemens, Tarrytown, NY); insulin levels were assessed using
CAAAGGACCAGCTGGACA, reverse: ATCGATGACAGCGCCTCA; IL‐6,
the commercial immunoassay kit (EZRMI‐13K #pipe, Rat/Mouse
forward: TCTCTGGGAAATCGTGGA, reverse: CTCCAGAAGACCAGA
Insulin ELISA; Merck, Millipore, Darmstadt, Germany) according to
GGA; IFN‐γ, forward: TGAACGCTACACACTGCATCTTGG, reverse: CGA
the manufacturer’s specifications.
CTCCTTTTCCGCTTCCTGAG; iNOS, forward: TTTGCTTCCATGCTAA TGCGAAAG, reverse: GCTCTGTTGAGGTCTAAAGGCTCCG; GAPDH,
2.2 | Sample preparation
forward: ACCCAGAAGACTGTGGATGG, reverse: CACATTGGGGGTA GGAACAC.
At the end of observation after harvesting, AT was carefully dissected. Dried intra‐abdominal AT depots (epididymal, retroperitoneal perirenal, mesenteric, omental) and subcutaneous AT depots (dorsal and inguinal)
2.5 | Statistical analysis
were weighed after drying excess of blood and tissue fluid using tissue
Statistical analyses were performed using SPSS 18.0 software (SPSS,
paper. The quantification was expressed as % of body weight. AT was
Inc., Chicago, IL). Data are presented as the mean ± standard
also processed for isolation of the stroma‐vascular fraction (SVF). Briefly,
deviation of the mean (SD). The comparisons between experimental
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3
groups were performed using an unpaired Student’s t test with
reduction in AT and particularly in the VAT depot. In the sildenafil
Welch’s correction. Pearson’s correlation coefficient was used to
group, compared with the vehicle, there was a significant reduction
measure any linear association between variables. A probability of
in AT. Total AT was 28.31 ± 2.18% in the db/db + vehicle group and
less than 5% (p < 0.05) or 1% (p < 0.01) was considered statistically
23.79 ± 3.34% in the db/db + sildenafil group (p = 0.004; Figure 1b).
significant.
This reduction may be attributed to the reduction in VAT, 18.90 ± 1.54% versus 15.94 ± 1.76% (p = 0.002; Figure 1c), rather than SAT, 9.41 ± 1.71% versus 7.85 ± 1.86% (p = 0.083; Figure 1d). When
3 | RES U LTS
we considered VAT and SAT in grams and correlated these with the mean change in body weight from baseline, we observed different
Compared with the vehicle, sildenafil‐treated mice showed a
correlations between the two treatments. A positive correlation
significant reduction in the body weight. Body weight before
was found between the mean change in body weight from baseline
treatment was 47.22 ± 4.02 g in the db/db + vehicle group and
and VAT, significant in the db/db + vehicle group (R = 0.924,
50.22 ± 2.39 g in the db/db + sildenafil group, p = 0.080; at the end
p < 0.001) and in the db/db + sildenafil group (R = 0.716, p = 0.004;
of the observation period, body weight was 52.67 ± 7.53 g in the
Figure 2a). SAT positively correlated with the mean change in body
db/db + vehicle group and 42.67 ± 6.6 g in the db/db + sildenafil
weight from baseline, but this correlation was significant only in
group, p = 0.010 (Figure 1a), but no change was observed in blood
the db/db + vehicle group (R = 0.789, p = 0.011), and not in the
glucose levels (Fiore, Gianfrilli, et al., 2016) or insulin levels
db/db + sildenafil group (R = 0.331, p = 0.105; Figure 2b), suggesting
(1.98 ± 0.81 ng/ml in the db/db+vehicle group and 1.77 ± 1.11 ng/ml
a major contribution of VAT toward the reduction in weight in
in the db/db + sildenafil group, p = ns). We found that the reduction
sildenafil‐treated mice. To better understand the cellular composi-
in body weight after sildenafil treatment was associated with the
tion of SAT under sildenafil treatment, we analyzed SVF of SAT and
(a)
Body weight (g)
80
db/db+Sildenafil
60
* 40
20
total fat % of body weight
(b) db/db+vehicle
35
* 30 25 20
db /d b+ ve hi cle
s
e
th
lin
(d)
22
*
20 18 16 14 12
15
10
5
db /d b+ Si ld en af il
db /d b+ Si ld en af il
db /d b+ ve hi cle
0
db /d b+ ve hi cle
VAT%of body weight
24
SAT % of body fat weight
+
3m
on
se ba
(c)
db /d b+ Si ld en af il
15 0
F I G U R E 1 (a) The plot represents mean ± SD body weight at baseline and after 3 months of treatments; (b) AT%, (c) VAT%, and (d) SAT% in vehicle‐treated (black) or sildenafil‐treated mice (gray). Results are expressed as mean ± SD. *p < 0.05; db/db + vehicle, n = 9; db/db + sildenafil, n = 9. AT, adipose tissue; VAT, visceral AT; SAT, subcutaneous AT
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(a)
15
db/db+vehicle
8
(b)
6 SAT (g)
VAT (g) 5
db/db+vehicle db/db+Sildenafil
db/db+Sildenafil 10
ET AL.
4
2
0 -20
-10
0
10
20
30
Body Weight Change form Baseline(g)
0 -20
-10
0
10
20
30
Body Weight Change form Baseline(g)
F I G U R E 2 Correlation between VAT (g) (a) and SAT (g) (b) with body weight change from baseline (g) in vehicle‐treated (black) or sildenafil‐ treated mice (gray); db/db + vehicle, n = 9; db/db + sildenafil, n = 9. VAT, visceral AT; SAT, subcutaneous AT
observed that sildenafil treatment led to an increase of F4/80+ +
Sildenafil treatment in humans could explain both the amelioration
macrophages M2 (db/db + vehicle vs. db/db + sildenafil:
of insulin resistance (Ramirez et al., 2015) and the modification of
16.89 ± 5.76 vs. 29.28 ± 9.47, p = 0.004; Figure 3a,b) and of
visceral depots, measured as waist circumference and epicardial
CD31+CD45−EC (db/db + vehicle vs. db/db + sildenafil: 2.57 ± 0.96
AT, without any changes in body weight (Fiore, Gianfrilli, et al.,
vs. 3.99 ± 0.70, p = 0.008; Figure 3c,d). APs, indicated as a
2016), suggesting an effect of AT remodeling. These results
Mrc1
+
−
−
(db/db + vehicle vs. db/db +
could strengthen the outstanding role of the cGMP/PDE5 signaling
sildenafil: 5.00 ± 1.55 vs. 5.99 ± 1.68, p = 0.300; Figure 3e,f), also
pathway in the homeostasis in the cardiovascular system
increased in sildenafil‐treated diabetic animals, although this was
(Giannetta et al., 2014, 2012; Mandosi et al., 2015; Pofi et al.,
not statistically significant. No significant difference was found in
2017; Venneri et al., 2015), acting as well as in the prevention
PDE5 expression (Figure 4a). Moreover, tissue cytokine expres-
of risk factors.
percentage of Sca1 CD31 CD45
sions of interleukin 10 (IL‐10), CCL2, inducible nitric oxide
The cellular characteristics of fat depots, including adipocyte size,
synthase (iNOS), IL‐6, and interferon‐γ (IFN‐γ) were significantly
macrophage accumulation, arteriolar dysfunction, angiogenesis, and
modified in the sildenafil AT, providing an apparent link between
cellular hypoxia, promote a state of chronic low‐grade inflammation,
morphological–functional changes in AT and tissue inflammation.
related to metabolic derangements (Fox et al., 2007).
Specifically, IL‐10 in the VAT db/db + vehicle group versus the
Regulation of the altered balance of macrophages and improve-
db/db + sildenafil group: 1.2 ± 0.6 versus 1.9 ± 0.45, p < 0.05, and in
ment of EC populations could be a further advanced approach to
SAT 1.5 ± 0.47 versus 2.1 ± 0.5, p < 0.05 (Figure 4b). CCL2 in VAT
ameliorate lipid and glucose metabolism and improve insulin
db/db + vehicle versus db/db + sildenafil: 1.85 ± 0.35 versus 0.38 ±
sensitivity. In this study, we confirmed in the SAT depot the results
0.02, p < 0.05, and in SAT 1.16 ± 0.67 versus 0.57 ± 0.02, p < 0.05
of sildenafil treatment found in VAT on macrophages and ECs (Fiore
(Figure 4c). INOS in VAT db/db + vehicle versus db/db + sildenafil:
et al., 2016). Additionally, we found that PDE5 activity is required
1.08 ± 0.37 versus 0.65 ± 0.18, p < 0.05 (Figure 4d). IL‐6 in SAT 1.01
for CCL2 and IFN‐γ expressions in AT, and sildenafil may be able to
± 0.18 versus 0.40 ± 0.15, p < 0.05 (Figure 4d). IFN‐γ in VAT
suppress chronic inflammation in obesity by inhibiting the expres-
db/db + vehicle versus db/db+sildenafil: 1.06 ± 0.38 versus 0.49 ±
sion of this inflammatory cytokine and promoting upregulation of
0.17, p < 0.05; in SAT 1.07 ± 0.43 versus 0.43 ± 0.25, p < 0.05
the anti‐inflammatory IL‐10 cytokine. Moderately increased IL‐6
(Figure 4f).
levels found in obese individuals may contribute toward the development of obesity‐associated complications, for example, insulin resistance, via the inhibition of lipoprotein lipase activity in
4 | D IS C U S S IO N
the adipose tissue (Scheller, Chalaris, Schmidt‐Arras, & Rose‐John, 2011). Our results indicate that adipose tissue isolated from SAT of
Several lines of experimental evidence suggest that the quality and
sildenafil‐treated mice expressed less IL‐6 than those from SAT of
distribution of AT influence the development of obesity‐associated
vehicle‐treated mice, indicating that VAT is less responsible for the
pathologies. The regional distribution of body fat appears to be
downregulation. This study provides further evidence of the
much more important than excess adiposity per se in driving the
involvement of PDE5 inhibition in preserving the quality of AT by
cardiovascular risk. Maintenance of SAT homeostasis has the
limiting AT inflammation and promoting healthier fat deposits, and
potential to become a next‐generation target for the treatment of
expands our understanding of the relationship between inflamma-
obesity and diabetes. In the current study, we provide data
tory signaling pathways and adipose tissue function.
supporting the role of PDE5 inhibition in body composition,
APs are functionally plastic cells that are influenced by
highlighting a predominant effect in reducing VAT rather than
their physiologic context. Recently, a study on a high‐fat diet
SAT. Preservation of SAT and reduction of VAT represent possible
rabbit model demonstrated that the PDE5i, tadalafil, promoted
mechanisms of metabolic protection. Redistribution of fat after
preadipocyte differentiation, improving AT (Maneschi et al., 2016).
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(a)
db/db+vehicle
db/db+sildenafil
5
(b)
Side Scatter
Mrc1+ % of F4/80 +cells
Gated on F4/80+ cells
50
**
40 30 20 10
Mrc-1
db /d b+ Si lde na fil
db /d b+ ve hi cle
0
(d)
(c)
6
CD31
CD31+ CD45-cells
** 4
2
CD45
(f)
10
5
0 db /d b+ Si lde na fil
Sca1
15
db /d b+ ve hi cle
Side Scatter
Sca1+% of CD45-CD31- cells
Gated on CD31- CD45- cells
(e)
db /d b+ Si lde na fil
db /d b+ ve hi cle
0
F I G U R E 3 (a) The FACS plots show representative gating of Mrc1+ cells, previously gated on F4/80+ cells in db/db + vehicle (left panel), db/db + sildenafil (right panel). (b) Quantification of percentage of Mrc1+F4/80+ cells, in vehicle‐treated (black) or sildenafil‐treated mice (gray). (c) Representative gating of CD31+CD45− cells in db/db + vehicle (left panel), db/db + sildenafil (right panel). (d) Quantification of CD31+CD45‐ endothelial cells, in vehicle‐treated (black) and sildenafil‐treated mice (gray). (e) Representative gating of Sca1+ cells on CD31−CD45− cells in db/db + vehicle, left panel; db/db + sildenafil, right panel. (f) Percentage of SCA1+CD45−CD31− adipocytes progenitor cells, in vehicle‐treated (black) or sildenafil‐treated mice (gray). Results are expressed as mean ± SD. **p < 0.01. CCL2: chemokine (C–C motif) ligand 2; IFN‐γ: interferon‐γ; IL‐6: interleukin 6; iNOS: inducible nitric oxide synthase; VAT: visceral adipose tissue
In VAT of obese diabetic mice, we observed that PDE5i led to a
significant hyperplasia in response to obesogenic stimuli (Wang,
significant increase in APs, which indicated a propensity to AT
Tao, Gupta, & Scherer, 2013).
hyperplasia rather than hypertrophy (Fiore, Gianfrilli, et al., 2016).
The use of PDE5–cGMP‐related pathways might contribute toward
In SAT, the minor increase in AP frequency could be due to the
defining the regulatory signals in AT microenvironments, and toward the
intrinsic characteristic of mouse SAT, which does not exhibit
design of novel approaches to control fat distribution in a manner that
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(a)
(b)
2.0
* 3.0
1.0 0.5
2.0 1.5 1.0 0.5
db /d b+ ve VA hi cle T db /d b+ Si ld en SA af il T db /d b+ ve SA hi cle T db /d b+ Si ld en af il
0.0
db /d b+ ve VA hi cle T db /d b+ Si ld en SA af il T db /d b+ ve SA hi cle T db /d b+ Si ld en af il
0.0
(d)
*
3.0
*
2.5
*
iNOS/GAPDH (fold change)
CCL2/GAPDH (fold change)
VA T
VA T
(c) 2.0
*
*
2.5 IL-10/GAPDH (fold change)
PDE5/GAPDH (fold change)
1.5
2.5
ET AL.
1.5 1.0
2.0
*
1.5 1.0 0.5
0.5
0.0
VA T
VA T
db /d b+ ve VA hi cle T db /d b+ Si ld en SA af il T db /d b+ ve SA hi cle T db /d b+ Si ld en af il
db /d b+ ve VA hi cle T db /d b+ Si ld en SA af il T db /d b+ ve SA hi cle T db /d b+ Si ld en af il
0.0
(e)
(f)
3.0
3.0
*
1.5 1.0
IFN /GAPDH (fold change)
2.0
2.5 2.0
1.0
0.0
0.0 db /d b+ ve VA hi cle T db /d b+ Si ld en SA af il T db /d b+ ve SA hi cle T db /d b+ Si ld en af il
0.5
db /d b+ ve VA hi cle T db /d b+ Si ld en SA af il T db /d b+ ve SA hi cle T db /d b+ Si ld en af il
*
1.5
0.5
VA T
* *
VA T
IL-6/GAPDH (fold change)
2.5
F I G U R E 4 Relative quantification of PDE5 (a), IL‐10 (b), CCL2 (c), iNOS (d), IL‐6 (e), IFN‐γ, and (f) gene expression ± SD in n = 5 of VAT db/db + vehicle, VAT db/db + sildenafil, SAT db/db + vehicle, SAT db/db + sildenafil db/db + vehicle, db/db + sildenafil groups. CCL2: chemokine (C–C motif) ligand 2; IFN‐γ, interferon‐γ; IL‐6, interleukin 6; iNOS, inducible nitric oxide synthase; PDE5, phosphodiesterase type 5; SAT, subcutaneous adipose tissue; VAT, visceral adipose tissue
favors metabolically beneficial SAT over detrimental VAT. Future studies are warranted for a more detailed understanding of the causal link between PDE5i and its pathways in promoting AT health, tightly connected with systemic metabolism.
AC KNO WL EDG EM E NT S The study was funded by the Ministry of University and Research (2015ZTT5KB).
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CO NFLICTS OF INTE RES T The authors declare that there are no conflicts of interest.
A U T H O R S’ CON TR IBU TI ONS D.F. designed and carried out experiments, analyzed the data, and wrote the manuscript; D.G. analyzed the data and wrote the manuscript; S.C analyzed the data; F.N. and A.L. discussed the data and critically revised the manuscript; A.M.I. conceived and designed experiments, provided financial support, and wrote the manuscript; and M.A.V. conceived, designed, and carried out experiments, directed the project, and wrote the manuscript.
E TH I C S A P P R O V A L A N D CO N S E N T TO P A R T I C I P A TE All experiments were performed in accordance with Italian law (D.L. 2010/63EU) and the study was approved by the Sapienza University’s Animal Research Ethics Committee and the Italian Ministry of Health (165/2016‐PR).
OR CID Fabio Naro
http://orcid.org/0000-0003-0572-5900
Andrea Lenzi
http://orcid.org/0000-0002-7711-0465
Andrea M. Isidori Mary A. Venneri
http://orcid.org/0000-0002-9037-5417 http://orcid.org/0000-0002-0687-8135
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How to cite this article: Fiore D, Gianfrilli D, Cardarelli S, et al. Chronic phosphodiesterase type 5 inhibition has beneficial effects on subcutaneous adipose tissue plasticity in type 2 diabetic mice. J Cell Physiol. 2018;1–7. https://doi.org/10.1002/jcp.26796
