OBJECTIVE: To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-α-induced apoptosis of brown adipocytes. DESIGN: 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-α and/or cycloheximide (CHX; 10 μg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and/or 10 μM heat shock protein 70 (HSP70) antisense or sense oligomers. MEASUREMENTS: 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-α 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. CONCLUSION: These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-α-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.
Nisoli, E., Regianini, L., Bulbarelli, A., Briscini, L., Breacale, R., Carruba, M. (2001). Protective effects of noradrenaline against tumor necrosis factor-α-induced apoptosis in cultured rat brown adipocytes: Role of nitric oxide-induced heat shock protein 70 expression. INTERNATIONAL JOURNAL OF OBESITY, 25(10), 1421-1430 [10.1038/sj.ijo.0801788].
Protective effects of noradrenaline against tumor necrosis factor-α-induced apoptosis in cultured rat brown adipocytes: Role of nitric oxide-induced heat shock protein 70 expression
Bulbarelli, A.;
2001
Abstract
OBJECTIVE: To elucidate the effects and molecular mechanism(s) by means of which noradrenaline (NA) protects against the tumor necrosis factor (TNF)-α-induced apoptosis of brown adipocytes. DESIGN: 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-α and/or cycloheximide (CHX; 10 μg/ml) and/or NA and/or nitric oxide (NO) donors and/or the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and/or 10 μM heat shock protein 70 (HSP70) antisense or sense oligomers. MEASUREMENTS: 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-α 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. CONCLUSION: These findings demonstrate that the NO produced by NA stimulation can induce resistance to the TNF-α-induced apoptosis of brown adipocytes, possibly by means of the expression of HSP70.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.