Protective effects of noradrenaline against tumor necrosis factor-alpha-induced apoptosis in cultured rat brown adipocytes: role of nitric oxide-induced heat shock protein 70 expression.
OBJECTIVE
To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-alpha-induced apoptosis of brown adipocytes.


METHODS
Brown fat precursor cells were isolated from young rats; 2.5 million cells were added to each 24-well culture plate and cultured in a defined culture medium. On day 8, the cultured cells were exposed to murine recombinant TNF-alpha and/or cycloheximide (CHX; 10 microg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and/or 10 microM heat shock protein 70 (HSP70) antisense or sense oligomers.


METHODS
Analysis of DNA fragmentation on agarose gel as a marker of apoptosis; reverse transcriptase-polymerase chain reaction analysis of mRNA levels; Western blotting analysis of protein levels.


RESULTS
Pretreatment of primary cultures of rat brown fat cells with micromolar concentrations of NA or the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) induced the expression of HSP70 mRNA and protein, which was associated with cytoprotection against TNF-alpha plus CHX-induced apoptosis. The L-NAME inhibitor of NO synthase activity inhibited both NA-stimulated HSP70 expression and cytoprotection. Furthermore, pretreatment of brown adipocytes with an antisense oligonucleotide to HSP70 antagonized both SNAP- and NA-induced cytoprotection.


CONCLUSIONS
These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-alpha-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.
Nisoli
E
E
Center for Study and Research on Obesity, Department of Preclinical Sciences, LITA Vialba, L Sacco Hospital, Milan University, Milan, Italy. enzo.nisoli@unimi.it
Regianini
L
L
Bulbarelli
A
A
Briscini
L
L
Bracale
R
R
Breacale
R
R
Carruba
M O
MO
D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D053938:DNA Fragmentation; D004587:Electrophoresis, Agar Gel; D018840:HSP70 Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D009569:Nitric Oxide / Q000494:pharmacology*; D009603:Nitroso Compounds; D009638:Norepinephrine / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011924:Rats, Zucker; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D018840:HSP70 Heat-Shock Proteins; D009603:Nitroso Compounds; D016376:Oligonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D003513:Cycloheximide; D009638:Norepinephrine