Introduction: We previously reported the novel finding that leptin, and several other fat-derived hormones (adipokines), is expressed and regulated in rat hypothalamus (1). Subsequently leptin (ob) mRNA was found in human, sheep and pig brain, but was surprisingly undetectable in mouse brain. In the present experiments we used a mouse hypothalamic neuronal cell line to investigate possible inhibitory mechanisms that may prevent mouse neurons from expressing ob mRNA. These included: (a) blockade of histone deacetylase (HDAC); (b) reversal of DNA methylation; (c) inhibition of protein synthesis.
Methods: Mouse N-1 hypothalamic neurons (2) were maintained in DMEM culture medium containing 10% FBS at 37 °C in 5% CO2/95% air. Cells were plated at 200,000 cells/well in 6-well culture plates and allowed to grow to confluence (48 hr). Cells were then treated with: (1) trichostatin A (TSA; HDAC inhibitor; 500 nM; 24 hr); (2) 5-aza-2-deoxycytidine (AZA; DNA methylation inhibitor; 5 μM; 48 hr); (3) protein synthesis inhibitors (cycloheximide, CHX, 25 μg/ml; puromycin, 50 μg/ml; anisomycin, 5 μM; 4 hr). Cells were harvested for preparation of total RNA and ob mRNA was quantified by realtime RT-PCR.
Results: Ob mRNA was undetectable in untreated mouse N-1 neurons. Blockade of histone deacetylation with TSA, or depletion of DNA methylation with AZA, had no effect on ob mRNA; i.e., leptin gene expression remained undetectable. In marked contrast, inhibition of protein synthesis with several inhibitors profoundly increased levels of ob mRNA. For example, CHX (25 μg/ml) induced the expression of ob mRNA by 60 min post-treatment and this effect reached a plateau at 7 hr. Half-maximal stimulation occurred at 2.5 μg/ml.
Conclusion: Under in vitro conditions chromatin modification, by histone acetylation, or DNA methylation, appears not to be implicated in the inability of mouse hypothalamic neurons to express the ob gene. However, ob mRNA levels are superinduced following protein synthesis inhibition. These data suggest the existence of an inhibitory protein that normally suppresses ob expression in mouse brain. Supported by IWK, UIMRF/Capital Health.
(1) M Wilkinson, RE Brown, SA Imran and E Ur (2007). Adipokine gene expression in brain and pituitary gland. Neuroendocrinology 86: 191209.
(2) Brown RE et al. (2007). Neuroendocrinology 85: 232241.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.