Effects of nitric oxide on proliferation and differentiation of rat brown adipocytes in primary cultures

1 In the present work, we study the effect of NO on the proliferation and differentiation of brown fat cells in primary cultures. 2 Brown fat precursor cells isolated from rat brown adipose tissue were cultured for 8 days until confluence and treated daily with the NO donating agents, S‐nitroso‐acetyl penicillamine (SNAP) or S‐nitroso‐ L ‐glutathione (GSNO). Both agents (300 μ M ) decreased cell proliferation approximately 8 fold on day 8. The inhibitory effect of NO was unlikely to be due to cytotoxicity since (i) cells never completely lost their proliferation capacity even after 8 days of exposure to repeated additions of SNAP or GSNO, and (ii) the inhibitory effect was reversible after removal of the media containing NO donors. 3 Daily treatment with nitric oxide synthase inhibitors, such as N G ‐nitro‐ L ‐arginine methyl ester ( L ‐NAME, 300 μ M ), led to the stimulation of cell proliferation by 44±5%, n =3, suggesting that NO, endogenously produced in brown adipocytes, may be involved in modulating cell growth. 4 Daily treatment with both SNAP or GSNO induced significant mitochondriogenesis, measured as the mitochondrial conversion of 3‐[4,5‐dimethylthiazol‐2‐yl‐]‐2,5‐diphenyl tetrazolium bromide (MTT) to formazan, whilst daily treatment with L ‐NAME was without effect. 5 The inhibition of cell proliferation by NO donors was accompanied by the expression of two genes coding for peroxisome proliferator activated receptor‐γ and uncoupling protein‐1, which are upregulated during differentiation. 6 Increasing cyclic GMP in cells by 8‐bromo‐cyclic GMP (100–1000 μ M ) did not reproduce the observed NO effects on either cell number or gene expression. On the other hand, chronic treatment with the inhibitor of the NO‐stimulated guanylyl cyclase, 1H‐[1,2,4]oxadiazole[4,3‐a]quinoxalin‐1‐one (ODQ), reduced the expression of peroxisome proliferator activated receptor‐γ and uncoupling protein‐1.British Journal of Pharmacology (1998) 125 , 888–894; doi: 10.1038/sj.bjp.0702131