Phosphodiesterase-5A and neutral endopeptidase activities in human adipocytes do not control atrial natriuretic peptide-mediated lipolysis.
OBJECTIVE
Atrial natriuretic peptide (ANP) stimulates lipolysis in human adipocyte through a cGMP signalling pathway, the regulation of which is poorly known. Since phosphodiesterases (PDE) and neutral endopeptidase (NEP) play a major role in the regulation of the biological effects of natriuretic peptides in the cardiovascular and renal systems, we investigated whether these mechanisms could regulate cGMP signalling and ANP-mediated lipolysis in human adipocytes.


METHODS
The presence of cGMP-specific PDE and NEP in differentiated pre-adipocytes and in mature adipocytes was evaluated by real-time qPCR and Western blot. The effect of non-selective and selective inhibition of these enzymes on ANP-mediated cGMP signalling and lipolysis was determined in isolated mature adipocytes.


RESULTS
PDE-5A was expressed in both pre-adipocytes and adipocytes. PDE-5A mRNA and protein levels decreased as pre-adipocytes differentiated (10 days). PDE-5A is rapidly activated in response to ANP stimulation and lowers intracellular cGMP levels. Its selective inhibition by sildenafil partly prevented the decline in cGMP levels. However, no changes in baseline- and ANP-mediated lipolysis were observed under PDE-5 blockade using various inhibitors. In addition, NEP mRNA and protein levels gradually increased during the time-course of pre-adipocyte differentiation. Thiorphan, a selective NEP inhibitor, completely abolished NEP activity in human adipocyte membranes but did not modify ANP-mediated lipolysis.


CONCLUSIONS
Functional PDE-5A and NEP activities were present in human adipocytes, however these enzymes did not play a major role in the regulation of ANP-mediated lipolysis.
Moro
C
C
INSERM, U858, Obesity Research Laboratory, Toulouse, France.
Klimcakova
E
E
Lafontan
M
M
Berlan
M
M
Galitzky
J
J
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D000328:Adult; D009320:Atrial Natriuretic Factor / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000096:biosynthesis / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D007700:Kinetics; D008066:Lipolysis / Q000187:drug effects*; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D058986:Phosphodiesterase 5 Inhibitors; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D015244:Thiorphan / Q000494:pharmacology; D013997:Time Factors
D058986:Phosphodiesterase 5 Inhibitors; D012333:RNA, Messenger; D009320:Atrial Natriuretic Factor; D015244:Thiorphan; D000242:Cyclic AMP; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D015260:Neprilysin; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine